KR101051385B1 - Primer set for obesity gene amplification, reagent for amplifying obesity gene comprising same and use thereof - Google Patents

Abstract

As a primer set for amplifying a target region including a target region of detection in an obesity gene (β2AR gene, β3AR gene and UCP1 gene) by a gene amplification method, a primer set capable of specifically amplifying the region is provided. to provide. Forward primer consisting of the nucleotide sequences of SEQ ID NO: 9 or SEQ ID NO: 109, SEQ ID NO: 25 and SEQ ID NO: 43, and reverse primer consisting of the nucleotide sequences of SEQ ID NO: 18, SEQ ID NO: 30, and SEQ ID NO: 63 Use three pairs of primer sets each containing. By using this primer set, it is possible to specifically amplify the target region including the site where the polymorphism for detection occurs in the β2AR gene, β3AR gene, and UCP1 gene in the same reaction solution.

Description

Primer set for obesity gene amplification, reagent for amplification of obesity gene comprising the same, and use thereof

The present invention relates to a primer set for amplifying the β2AR gene, the β3AR gene, and the UCP1 gene, which are obesity genes, a reagent for amplifying obesity genes including the same, and uses thereof.

As a gene polymorphism involved in obesity, a gene encoding β-2 adrenergic receptor (β2AR), a gene encoding β-3 adrenergic receptor (β3AR) (β3AR gene) and a deconjugated protein (UCP) are coded Polymorphism of the gene (UCP1 gene) is known. β2AR is mainly distributed in the heart, bronchial smooth muscle and the like, but also exists in adipose tissue and is involved in lipolysis. In the β2AR gene encoding this, there is a polymorphism (Arg16Gly) in which arginine (Arg) at amino acid position 16 is glycine (Gly). It is reported that the subject having this polymorphism (16% present in Japanese) has an increased metabolic rate at rest compared to the subject having no polymorphism. In addition, β3AR is present in brown adipose tissue and white adipose cell tissue, and by the binding of noradrenaline, heat production occurs in brown adipose tissue and lipolysis in white adipose tissue. In the β3AR gene encoding this, there is a polymorphism (Trp64Arg) in which tryptophan (Trp) at amino acid position 64 becomes arginine (Arg). It is reported that the subjects with this polymorphism (34% present in Japanese) have a decreased metabolic amount at rest compared to the subjects without the polymorphism. UCP1 is also a protein that plays a central role in heat production in brown adipose tissue when the exchange nerve is in an excited state. In the UCP1 gene encoding this, there is a polymorphism in which adenine (A) is -guanine (G) at -3826 position of mRNA (upper 3826 position of translation start point of UCP1 gene in genomic sequence). It is reported that the subjects with this polymorphism (24% present in Japanese obese women) have a decreased metabolic rate in the whole body compared to the subjects without the polymorphism. From the relationship between the polymorphism and metabolic amount of each gene as described above, it is actually performed to analyze the polymorphism of these genes and use the analysis result for the treatment and prevention of obesity of the subject.

On the other hand, as a method of analyzing the causes of all diseases, the ease of disease disease (easiness of disease) between the individuals, the difference in drug efficacy among individuals, etc., point mutations, so-called monobasic polymorphisms (SNP) Is widely detected. As a general detection method of point mutations, (1) a direct sequencing method in which a region corresponding to a sequence to be detected is amplified by PCR (Polymerase chain reaction) on a target DNA of a sample, and the entire gene sequence is analyzed (2 A) A region corresponding to the sequence to be detected is amplified by PCR with respect to the target DNA of the sample, and the amplification product is cleaved by a restriction enzyme having a different cleavage effect due to the presence or absence of a target mutation in the sequence to be detected. RFLP analysis, which performs typing by electrophoresis, and (3) ASP-PCR method which performs PCR using primers in which the target mutation is located in the 3 'terminal region, and determines the mutation by the presence or absence of amplification. .

However, these methods require time and cost, for example, since purification of the DNA extracted from a sample, electrophoresis, and restriction enzyme treatment are essential. Moreover, since it is necessary to open a reaction container once after PCR, the said amplification product may be mixed in the next reaction system, and there exists a possibility that analysis accuracy may fall. In addition, since automation is difficult, a large amount of samples cannot be analyzed. Moreover, about the ASP-PCR method of said (3), there also exists a problem that specificity is low.

In view of such a problem, a method of analyzing the melting temperature (Tm: melting temperature) of a double-stranded nucleic acid formed from a target nucleic acid and a probe has been put into practical use as a method for detecting a point mutation. Such a method is called a melting curve analysis because it is performed by Tm analysis or analysis of the said double chain melting curve, for example. This is the following method. That is, first, a hybrid (double-stranded DNA) of the target single-stranded DNA of the detection sample and the probe is formed using a probe complementary to the detection target sequence including the point mutation for detection. Subsequently, heat processing is performed on this hybrid formation body, and the dissociation (fusion) of a hybrid accompanying a temperature rise is detected by the change of signals, such as absorbance. Then, the Tm value is determined based on the detection result to determine the presence or absence of the point mutation. The Tm value is higher as the homology of the hybrid body is higher, and lower as the homology is lower. For this reason, the Tm value (evaluation reference value) is calculated | required beforehand about the hybrid body of the detection object sequence containing a point mutation and the probe complementary to it, and the Tm value (target measurement) of the target single-stranded DNA of a detection sample is measured Value). If the measured value is equal to the evaluation reference value, it may be determined that there is a point mutation in the match, that is, the target DNA, and if the measured value is lower than the evaluation reference value, it is determined that there is no mismatch, that is, there is no point mutation in the target DNA. can do. And according to this method, automation is also possible.

However, also in the detection method using such a Tm analysis, there exists a problem that PCR must amplify specifically and efficiently the area | region containing a detection target site | part. In particular, many isozymes exist in the β2AR gene, the β3AR gene, and the UCP1 gene, which are important among the obesity genes, and the sequences encoding them are very similar. For this reason, in PCR, in addition to these genes of interest, there is a possibility that even the isozyme code gene is amplified. In addition, in the case of amplifying even other isozyme code genes in this way, for example, in the analysis of each of the polymorphisms of the β2AR gene, the β3AR gene, and the UCP1 gene (Non-Patent Documents 1 to 3), there is a cause of lowering the reliability of the analysis result. do. As described above, since a lot of effort is required to analyze one sample, there is also a problem that it is not practical to analyze a large amount of samples.

Non Patent Literature 1: PMID: 9399966 J Clin Invest. 1997 Dec 15; 100 (12): 3184-8.

Non Patent Literature 2: PMID: 9049481 Diabetologia. 1997 Feb; 40 (2): 200-4.

Non-Patent Document 3: PMID: 9541178 Diabetologia. 1998 Mar; 41 (3): 357-61.

Therefore, an object of the present invention is to provide a primer set for specifically amplifying a target region of at least one obesity gene selected from the group consisting of β2AR gene, β3AR gene and UCP1 gene by gene amplification.

In order to achieve the above object, the primer set of the present invention is a primer set for amplifying the obesity gene by a gene amplification method, the obesity gene is at least one selected from the group consisting of β2AR gene, β3AR gene and UCP1 gene It is a gene, It is characterized by including the at least 1 primer set selected from the group which consists of the following primer sets (1)-(3).

Primer Set (1)

A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F1) and a reverse primer consisting of an oligonucleotide of (R1)

(F1) at least one oligonucleotide having the same sequence as the region from the 4248th thymine base (T) in the nucleotide sequence of SEQ ID NO: 1 to the 18-25 base in the 5 'direction, as the first base; An oligonucleotide having the thymine base (T) at the 3 'end, and

At least one oligonucleotide having the same sequence as the region from the 18th to the 26th base in the 5 'direction with the 4250th thymine base (T) as the first base in the nucleotide sequence of SEQ ID NO: 1; Oligonucleotides with 3 'Terminus (T)

At least one oligonucleotide

(R1) at least one oligonucleotide complementary to a region from the 21st to 31th base in the 3 'direction with the 4292th adenine base (A) in the nucleotide sequence of SEQ ID NO: 1 as the first base; An oligonucleotide having a 3 'end of thymine base (T) complementary to the 4292th adenine base (A), and

At least one oligonucleotide complementary to the region from the 18th to the 27th base in the 3 'direction with the 4301th adenine base (A) in the nucleotide sequence of SEQ ID NO: 1 as the first base, wherein the 4301st Oligonucleotide having 3 'end of thymine base (T) complementary to adenine base (A)

At least one oligonucleotide

Primer Set (2)

A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F2) and a reverse primer consisting of an oligonucleotide of (R2)

(F2) at least one oligonucleotide having the same sequence as the region from the 4110's cytosine base (C) in the nucleotide sequence of SEQ ID NO: 2 as the 16th to 20th base in the 5 'direction, as the 1st base; Oligonucleotide having said cytosine base (C) as 3 'end

(R2) at least one oligonucleotide complementary to a region from the 15th to 19th base in the 3 'direction with the 4172nd guanine base (G) in the nucleotide sequence of SEQ ID NO: 2 as the first base; Oligonucleotide having 3 'end of cytosine base (C) complementary to 4,172th guanine base (G)

Primer Set (3)

A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F3) and a reverse primer consisting of an oligonucleotide of (R3)

(F3) at least one oligonucleotide having the same sequence as the region from the 25th to the 44th base in the 5 'direction with the 280th cytosine base (C) as the first base in the nucleotide sequence of SEQ ID NO: 3, Oligonucleotide having said cytosine base (C) as 3 'end

(R3) at least one oligonucleotide complementary to the region from the 23rd to the 38th base in the 3 'direction with the 350th cytosine base (C) as the first base in the nucleotide sequence of SEQ ID NO: 3, Oligonucleotide having 3 'end of guanine base (G) complementary to 350th cytosine base (C)

The reagent for amplifying a gene of the present invention is a reagent for amplifying an obesity gene by a gene amplification method, wherein the obesity gene is at least one gene selected from the group consisting of β2AR gene, β3AR gene, and UCP1 gene. It is characterized in that it comprises a primer set for obesity gene amplification of the present invention.

The method for producing an amplification product of the present invention is a method for producing an amplification product of an obesity gene by a gene amplification method, wherein the obesity gene is at least one gene selected from the group consisting of β2AR gene, β3AR gene, and UCP1 gene, It is characterized by including the following (I) process.

(I) At least one gene selected from the group consisting of the above β2AR gene, β3AR gene and UCP1 gene in the reaction solution, using the nucleic acid in the sample as a template and using the primer set for amplification of the obesity gene of the present invention. Process of amplifying

The polymorphism analysis method of the present invention is a method for analyzing a polymorphism of a detection target site in an obesity gene, wherein the obesity gene is at least one gene selected from the group consisting of β2AR gene, β3AR gene, and UCP1 gene. (Iii) to (iii).

(Iii) amplifying a region in the reaction solution containing a region to be detected in at least one gene selected from the group consisting of the β2AR gene, the β3AR gene, and the UCP1 gene by the method for producing an amplification product of the present invention

(Ii) a probe capable of hybridizing to an amplification product in the step (iii) and a detection target site in at least one gene selected from the group consisting of the β2AR gene, the β3AR gene, and the UCP1 gene. Process of preparing the reaction solution

(Iii) changing a temperature of the reaction solution and measuring a signal value indicating a state of melting of the hybridization product between the amplification product and the probe

(Iii) determining the polymorphism of the detection target site in at least one gene selected from the group consisting of the β2AR gene, the β3AR gene and the UCP1 gene from the change in the signal value according to the temperature change.

According to the primer set of the present invention, a target region including a site where a polymorphism for detection occurs in at least one obesity gene selected from the group consisting of the β2AR gene, the β3AR gene, and the UCP1 gene is specific in the reaction solution. It can also be amplified with high efficiency. For this reason, unlike the conventional method mentioned above, time and cost can be reduced. In this way, since the region including the detection target site where the specific polymorphism of the obesity gene occurs can be specifically amplified, for example, a probe complementary to the detection target sequence including the detection target site is also provided. By using it, it becomes possible to perform Tm analysis as it is using the said reaction liquid and to type the said polymorph. In addition, since amplification and typing can be performed with one reaction solution, the operation can be automated. In addition, when the primer set of the present invention is used, even if a sample containing a contaminant (for example, whole blood, oral mucosa, etc.) can be omitted, the amplification reaction can be performed more quickly and simply. Can be. In addition, when the primer set of the present invention is used, the amplification reaction can be performed at an amplification efficiency superior to that of the prior art, so that the amplification reaction can also be shortened. Therefore, according to the primer set of the present invention, a reagent containing the same, and a method for producing an amplification product using the same and a method for analyzing polymorphism, the polymorphism of at least one obesity gene selected from the group consisting of the β2AR gene, the β3AR gene, and the UCP1 gene can be quickly determined. It can be said to be very effective in the medical field because it can be interpreted simply and easily.

1 is a graph showing the results of a Tm analysis in Example 1 of the present invention.

It is a graph which shows the result of Tm analysis in the said Example 1 of this invention.

It is a graph which shows the result of Tm analysis in the said Example 1 of this invention.

It is a graph which shows the result of Tm analysis in Example 2 of this invention.

It is a graph which shows the result of Tm analysis in Example 3 of this invention.

It is a graph which shows the result of Tm analysis in a comparative example.

It is a graph which shows the result of Tm analysis in Example 4 of this invention.

<Primer set for obesity gene amplification>

Obesity gene amplification primer set of the present invention, as described above, characterized in that it comprises at least one primer set selected from the group consisting of the primer set (1) to (3). By including at least one primer set, it is possible to specifically amplify a specific target region in at least one obesity gene selected from the group consisting of, for example, a β2AR gene, a β3AR gene, and a UCP1 gene.

The primer set for the obesity gene amplification of the present invention may include only one kind of the primer sets (1) to (3), or may include any two kinds or both of the primer sets (1) to (3). good. As will be described later, the target region that can be specifically amplified by the primer set (1) is a region including a site (4265th in SEQ ID NO: 1) in which a specific polymorphism of the β2AR gene occurs, and the primer set ( The target region which can be specifically amplified by 2) is a region including the site (4134th in SEQ ID NO: 2) where a specific polymorphism of the β3AR gene occurs, and is specific by primer set (3). The target region that can be amplified by is a region including the site (292 th in SEQ ID NO: 3) where a specific polymorphism of the UCP1 gene occurs.

As described above, the specific polymorphisms of the β2AR gene, the β3AR gene, and the UCP1 gene, which are obesity genes, are all known as polymorphisms that affect metabolism. For this reason, it is important to investigate not only the polymorphism of any one type of gene but also the polymorphism of any two types of genes or all three types of genes. However, the conventional method has a problem in that a plurality of sequences cannot be analyzed in one reaction system. As described above, since many isozymes exist in each of the above-mentioned obesity genes, it is considered that the cause is also amplified in a code gene of isozyme other than the above-mentioned genes in PCR. Therefore, in order to examine the polymorphism of two types of genes or all three types of genes among the β2AR gene, the β3AR gene, and the UCP1 gene, the regions including the sites where the respective polymorphisms occur are respectively determined in separate reaction systems. It is necessary to interpret the amplification products obtained by amplification separately. As described above, in the conventional method, only two or three types of target regions each including a site where the above-mentioned polymorphism occurs in each gene are used as a template among the obesity genes. Amplification is very difficult. As described above, since much effort is required to analyze one sample, there is a problem that it is not practical to analyze a large amount of samples. On the other hand, according to the primer set for the obesity gene amplification of the present invention, even if any two or all three types of the primer sets (1) to (3) are included, the respective target regions are in the same reaction solution. At the same time it can also amplify specifically. For this reason, unlike the conventional method mentioned above, time and cost can be reduced. In addition, since two or three target regions are specifically amplified in the same reaction solution as described above, for example, by using a probe complementary to the detection target sequence in each target region, The Tm analysis can be performed as it is and the two or three types of polymorphs can be typed respectively. As described above, since the specific polymorphism in the two or three genes among the β2AR gene, the β3AR gene, and the UCP1 gene can be interpreted as the same reaction solution, the primer set for the obesity gene amplification of the present invention is, for example, It is also preferable to include any two or three types as well as the case of including any one of primer sets (1) to (3). Using such a set of obesity gene amplification primers, amplification of not only one target region but also two or three target regions at the same time enables efficient polymorphism analysis of the obesity gene.

In addition, below, a forward primer may be called F primer and a reverse primer may be called R primer.

First, the primer set 1 for amplifying the β2AR gene will be described. As described above, the primer set (1) is a pair of primer sets including a forward primer consisting of an oligonucleotide of the following (F1) and a reverse primer consisting of an oligonucleotide of the following (R1).

(F1) at least one oligonucleotide having the same sequence as the region from the 4250th thymine base (T) in the nucleotide sequence of SEQ ID NO: 1 to the 18-25 base in the 5 'direction, as the first base; An oligonucleotide having the thymine base (T) at the 3 'end, and

At least one oligonucleotide having the same sequence as the region from the 18th to the 26th base in the 5 'direction with the 4247th thymine base (T) as the first base in the nucleotide sequence of SEQ ID NO: 1, wherein the thymine base An oligonucleotide of at least one of the oligonucleotides having 3 'terminus (T)

(R1) at least one oligonucleotide complementary to the region from 21 to 31 bases in the 3 'direction with the 4292th adenine base (A) in the nucleotide sequence of SEQ ID NO: 1 as the first base, An oligonucleotide having a 3 'end of thymine base (T) complementary to the 4292th adenine base (A), and

At least one oligonucleotide complementary to the region from the 18th to the 27th base in the 3 'direction with the 4301th adenine base (A) in the nucleotide sequence of SEQ ID NO: 1 as the first base, wherein the 4301st Oligonucleotide having 3 'end of thymine base (T) complementary to adenine base (A)

At least one oligonucleotide

The nucleotide sequence shown in SEQ ID NO: 1 is the full length sequence of the ADRβ2 gene encoding a human (Homo sapiens) -derived β2 adrenergic, beta-2-, receptor, surface; ADRβ2 (β2AR), for example NCBI access: Registered in N0.DQ094845.

Hereinafter, this primer set (1) is also called "the primer set for (beta) 2AR gene." The primer set for the β2AR gene is a DNA chain including the 4249th to 4291th regions, the 4249th to 4300th regions, the 4251 to 4291th regions, or the 4251 to 4300th regions in SEQ ID NO: 1. And primer sets for amplifying the complementary chains thereof. The presence of a point mutation is known for the 4265th base (4265th base in SEQ ID NO: 1) in this region (4265A, 4265G). If the 4265th base is A, when the β2AR gene is translated into protein, the amino acid position 16 is arginine (Arg), and when the 4265th base is mutated to G, the amino acid position 16 is glycine (Gly). Indicates. In addition, these polymorphisms can be represented by 4265A / 4265A, 4265G / 4265G, and 4265A / 4265G in the case of a heteroconjugate in the case of a homoconjugate. In addition, when substituted with an amino acid, 4265A / 4265A may be represented by Arg-16 / Arg-16, 4265G / 4265G, Gly-16 / Gly-16 and 4265A / 4265G by Arg-16 / Gly-16. The notation by amino acid is the general notation of the polymorphism of the target β2AR gene. In addition, when only the said polymorphism of (beta) 2AR gene is analyzed, only the primer set for (beta) 2AR gene should be used.

In the present invention, the F1 primer and the R1 primer of the primer set for the β2AR gene need only have a base at the 3 'terminal that plays a role of determining the initiation point of amplification by a DNA polymerase. . By fixing the base at the 3 'end of each primer in this way, it is possible to sufficiently prevent the primer set for the β2AR gene from binding to a gene of similar other isozyme (for example, β1AR gene or the like).

Thus, since the base of the 3 'terminal should just be fixed for F1 primer and R1 primer, the length itself of each primer is not specifically limited, It can adjust suitably to general length. As an example of the length of a primer, it is the range of 13-50 mer, for example, Preferably it is 14-45 mer, More preferably, it is 15-40 mer. As a specific example, the F1 primer has the 4248th thymine base (T) in the nucleotide sequence of SEQ ID NO: 1 as the first base, and is in the 18-25 base (preferably 19 to 24) toward the 5 'direction. More preferably, at least one oligonucleotide having the same sequence as the region from the 19th to 23rd base) or the 4250th thymine base (T) in the nucleotide sequence of SEQ ID NO: 1 is used as the first base. It is preferable that it is at least 1 oligonucleotide which is the same sequence as the area | region up to 18-26 base (preferably 19-24 base, more preferably 19-22 base) toward 5 'direction. Further, the R1 primer is the 21-31st base (preferably the 22-30th base) toward the 3 'direction using the 4292th adenine base (A) in the base sequence of SEQ ID NO: 1 as the first base. More preferably at least one oligonucleotide complementary to the 23 to 28 base). In addition, since the 3 'ends of the F1 primer and the R1 primer are fixed, the region extending from the primer is, for example, the 4249 th to 4291 th regions and the 4249 th to 4300 th regions in SEQ ID NO: 1 as described above. It becomes an area | region, the 4251st-4291th area | region, or the 4251-4300th area | region, but the length of the whole amplification product obtained changes with the length of the primer to be used.

In addition, the R1 primer may not be an oligonucleotide that is completely complementary to the nucleotide sequence shown in SEQ ID NO: 1, and that the F1 primer is completely complementary to the nucleotide sequence. That is, in the part excluding the base at the 3 'end in each primer, the oligonucleotide which is completely complementary may be different from 1 to 5 bases.

The oligonucleotide constituting the R1 primer may be any of cytosine (C) and guanine (G) as the base complementary to the 4298th sequence in the nucleotide sequence of SEQ ID NO: 1. More preferably, as the R1 primer, it is preferable to simultaneously use both an oligonucleotide whose base is complementary at 4298 and an oligonucleotide whose base is complementary at 4298 is guanine. Polymorphs (4298C, 4298G) are known for the 4298th base in the nucleotide sequence of SEQ ID NO: 1, but the present invention is not intended to detect the polymorphism in this portion. For this reason, by using both oligonucleotides as R1 primers, any of 4298C and 4298G can be used for gene amplification by the above primers. It can be avoided sufficiently. The ratio of the oligonucleotide whose base is complementary at 4298 to cytosine and the oligonucleotide whose base is complementary to 4298 is not particularly limited. For example, a molar ratio of 1:10 to 10: 1 is preferred.

Although the specific example of F1 primer and R1 primer is shown below, this invention is not limited to this. "S" in the nucleotide sequences of SEQ ID NOs: 12 to 22 represents cytosine (C) or guanine (G) and corresponds to the base complementary to the 4298th sequence of SEQ ID NO: 1. In addition, the combination of these F1 primers and R1 primers is not limited at all, Among them, the F1 'primer consisting of oligonucleotides of SEQ ID NO: 9 or SEQ ID NO: 109 and oligos of SEQ ID NO: 12, SEQ ID NO: 18 or SEQ ID NO: 134 Particular preference is given to primer sets (1 ') comprising an R1' primer consisting of nucleotides. In addition, it is preferable that the R1 'primer which consists of oligonucleotides of SEQ ID NO: 12 and SEQ ID NO: 18 include both an oligonucleotide whose "s" is cytosine and an oligonucleotide whose "s" is guanine, as described above. . "Tm (degreeC)" in the following table | surface is Tm (degreeC) in the case where the sequence completely complementary with the sequence of the following table | surface is Tm (degreeC), and it is a paramater by MELTCALC software (http://www.meltcalc.com/). The value was calculated by setting the meter to an oligonucleotide concentration of 0.2 µM and sodium equivalent weight (Na eq.) 50 mM (hereinafter, the same). The Tm value can be calculated, for example, by conventionally known MELTCALC software (http://www.meltcalc.com/) or the like, and can also be determined by the nearest neighbor method (hereinafter, the same). . In the following table | surface, as an example of R1 primer, Tm value at the time of making guanine the base "s" complementary to the 4298th base of sequence number 1 is described.

Next, a primer set (2) for amplifying the β3AR gene will be described. As described above, the primer set (2) is a pair of primer sets including a forward primer consisting of an oligonucleotide of (F2) and a reverse primer consisting of an oligonucleotide of (R2).

(F2) at least one oligonucleotide having the same sequence as the region from the 4110's cytosine base (C) in the nucleotide sequence of SEQ ID NO: 2 as the 16th to 20th base in the 5 'direction, as the 1st base; Oligonucleotide having said cytosine base (C) as 3 'end

(R2) at least one oligonucleotide complementary to a region from the 15th to 19th base in the 3 'direction with the 4172nd guanine base (G) in the nucleotide sequence of SEQ ID NO: 2 as the first base; Oligonucleotide having 3 'end of cytosine base (C) complementary to 4,172th guanine base (G)

The nucleotide sequence shown in SEQ ID NO: 2 is the full length sequence of the ADRβ2 gene encoding a human derived β3 adrenergic receptor protein (Homo sapiens adrenergic, beta-3-, receptor: ADRβ3 (β3AR)), for example, NCBI accession: NO It is registered in .DQ104441.

Hereinafter, this primer set (2) is also called "the primer set for (beta) 3AR gene." The primer set for the β3AR gene is a primer set for amplifying a DNA chain including the 4111st-4141st region in SEQ ID NO: 2 and its complementary chain. The presence of a point mutation is known for the 4134th base (4134th base in SEQ ID NO: 2) in this region (4134T, 4134C). When the 4134th base is T, when the β2AR gene is translated into protein, the 64th amino acid is tryptophan (Trp), and when the 4134th base is changed to C, the 64th amino acid is arginine (Arg). Polymorphism. In addition, these polymorphisms can be represented by 4134T / 4134T or 4134C / 4134C in the case of a homo conjugate, and 4134T / 4134C in the case of a hetero conjugate. In addition, when substituted with an amino acid, 4134T / 4134T can represent Trp-64 / Trp-64, 4134C / 4134C can be represented by Arg-64 / Arg-64, 4134T / 4134C can be represented by Trp-64 / Arg-64, The notation with this amino acid is the general notation of the polymorphism of the target (beta) 3AR gene. When only the polymorphism of the β3AR gene is to be analyzed, only the primer set for the β3AR gene may be used.

As for the F2 primer and the R2 primer of the primer set for the β3AR gene, the base at the 3 'end, which serves to determine the start point of the amplification by the DNA polymerase, may satisfy the above conditions. By fixing the base at the 3 'end of each primer in this manner, it is possible to sufficiently prevent the primer set for the β3AR gene from binding to a gene of similar other isozyme (for example, β1AR gene or the like).

Since the F2 primer and the R2 primer are the same as the primer set for the β2AR gene, the base at the 3 'end, which serves to determine the initiation point of amplification by the DNA polymerase, may satisfy the above conditions. . For this reason, the length itself of F2 primer and R2 primer is not specifically limited, The same length as the above can be illustrated. As a specific example, the F2 primer has the 4110 th cytosine base (C) in the nucleotide sequence of SEQ ID NO: 2 as the first base, and the 16 to 20 base (preferably 17 to 20) toward the 5 'direction. It is preferable that it is at least 1 oligonucleotide which is the same sequence as the base and, more preferably, the 17th to 19th base). The R2 primer is the 15-19 base (preferably 16-19 base) toward the 3 'direction using the 4172nd guanine base (G) in the base sequence of SEQ ID NO: 2 as the first base. More preferably at least one oligonucleotide complementary to the region from bases 16 to 18). In addition, since the 3 'ends of the F2 primer and the R2 primer are fixed, the region extending from the primer is, for example, the 4111 to 4171th region in SEQ ID NO: 2 as described above, but the entire amplification product obtained is The length changes depending on the length of the primer to be used.

The R2 primer may not be an oligonucleotide that is completely complementary to the nucleotide sequence shown in SEQ ID NO: 2, and that the F2 primer is completely complementary to the nucleotide sequence described above. That is, in the part excluding the base at the 3 'end in each primer, the oligonucleotide which is completely complementary may be different from 1 to 5 bases.

Although the specific example of an F2 primer and an R2 primer is shown below, this invention is not limited to this. The combination of these F2 primers and R2 primers is not limited at all, but among them, the F2 'primer consisting of an oligonucleotide of SEQ ID NO: 24 or SEQ ID NO: 25 and the R2' consisting of an oligonucleotide of SEQ ID NO: 28 or SEQ ID NO: 30 Particular preference is given to primer sets (2 ') comprising primers.

Next, a primer set (3) for amplifying the UCP1 gene will be described. As described above, the primer set (3) is a pair of primer sets including a forward primer consisting of an oligonucleotide of the following (F3) and a reverse primer consisting of an oligonucleotide of the following (R3).

(F3) at least one oligonucleotide having the same sequence as the region from the 25th to the 44th base in the 5 'direction with the 280th cytosine base (C) as the first base in the nucleotide sequence of SEQ ID NO: 3, Oligonucleotide having said cytosine base (C) as 3 'end

(R3) at least one oligonucleotide complementary to the region from the 23rd to the 38th base in the 3 'direction with the 350th cytosine base (C) as the first base in the nucleotide sequence of SEQ ID NO: 3, Oligonucleotide having 3 'end of guanine base (G) complementary to 350th cytosine base (C)

The nucleotide sequence shown in SEQ ID NO: 3 is a gene sequence encoding a human polymorphic region 5 'of uncoupling protein (UCP), for example, registered in NCBI Accession: N0.U28479. have.

Hereinafter, the said primer set (3) is also called "the primer set for UCP1 gene." The primer set for the UCP1 gene is a primer set for amplifying a DNA strand comprising the 281st to 349th regions in SEQ ID NO: 3 and its complementary strand. The presence of a point mutation is known for the 292th base in this region (the 292th base in SEQ ID NO: 3) (292A, 292G). These polymorphisms can be represented by 292A / 292A and 292G / 292G in the case of homoconjugates and 292A / 292G in the case of heteroconjugates. The 292th base in SEQ ID NO: 3 corresponds to the -3826th base (upstream -3826th upstream of the UCP1 gene translation start point) in the mRNA of UCP1, and the aforementioned polymorphism is usually -3826A. / -3826A, -3826G / -3826G, -3826A / -3826G. When only the polymorphism of the UCP1 gene is to be analyzed, only the primer set for the UCP1 gene may be used.

As for F3 primer and R3 primer, the base of a 3 'terminal which plays a role of determining the starting point of amplification by a DNA polymerase should just satisfy the conditions mentioned above. By fixing the base at the 3 'end of each primer in this manner, the primer set for the UCP1 gene can be sufficiently prevented from binding to a gene of similar other isozyme (for example, UCP2, UCP3, UCP4 gene, etc.).

The F3 primer and the R3 primer of the primer set for the UCP1 gene are the same as the base set at the 3 'end, which serves to determine the initiation point of amplification by the DNA polymerase for the same reason as the primer set for the β2AR gene. You just need to satisfy. For this reason, the length itself of a F3 primer and an R3 primer is not specifically limited, The length same as the above can be illustrated. As a specific example, the F3 primer has the 280th cytosine base (C) in the nucleotide sequence of SEQ ID NO: 3 as the first base, and the 25 to 44 base (preferably 30 to 40) toward the 5 'direction. It is preferable that it is at least 1 oligonucleotide which is the same sequence as the base, More preferably, the area | region up to 32-37 base. In addition, the R3 primer has the 350th cytosine base (C) in the nucleotide sequence of SEQ ID NO: 3 as the first base, and the 23 to 38 base (preferably the 25 to 36 base) toward the 3 'direction. More preferably at least one oligonucleotide complementary to the region from bases 26 to 32). Since the 3 'ends of the F3 primer and the R3 primer are fixed, the region extending from the primer is, for example, the 281st to 349th regions in SEQ ID NO: 3, as described above. The whole length changes with the length of the primer to be used.

The R3 primer may not be an oligonucleotide that is completely complementary to the nucleotide sequence shown in SEQ ID NO: 3, and that the F3 primer is completely complementary to the nucleotide sequence described above. That is, in the part excluding the base at the 3 'end in each primer, the oligonucleotide which is completely complementary may be different from 1 to 5 bases.

The oligonucleotide constituting the R3 primer may be any of guanine (G) and thymine (T) with a base complementary to the 376th position in the nucleotide sequence of SEQ ID NO: 3. More preferably, as the R1 primer, it is preferable to simultaneously use both an oligonucleotide having a base complementary to 376th guanine and an oligonucleotide having a base complementary to 376th thymine. Polymorphs (376C, 376A) are known for the 376th base in the nucleotide sequence of SEQ ID NO: 3, but the present invention is not intended to detect the polymorphism in this portion. Therefore, by using both oligonucleotides as R3 primers, any of 376C and 376A can be amplified by the primers, and it is confirmed that amplification of the target region is affected by polymorphisms other than the detection purpose. It can be avoided sufficiently. Although the ratio of the oligonucleotide whose base complementary to 376th is guanine and the oligonucleotide whose base complementary to 376th is thymine is not specifically limited, For example, molar ratio 1: 10-10: 1 are preferable.

Although the specific example of F3 primer and R3 primer is shown below, this invention is not limited to this. "K" in the nucleotide sequences of SEQ ID NOs: 53 to 64 represents guanine (G) or thymine (T), and in SEQ ID NOs: 53 to 64 in the following table, complementary to the 376th base of SEQ ID NO: 3 Each Tm value in the case where the base is guanine or thymine is described. The combination of these F3 primers and R3 primers is not limited at all, but among these, a primer set (3 ') comprising an F2' primer consisting of an oligonucleotide of SEQ ID NO: 43 and an R3 'primer consisting of an oligonucleotide of SEQ ID NO: 63 Is particularly preferred. As described above, the R3 'primer composed of the oligonucleotide of SEQ ID NO: 63 preferably includes both an oligonucleotide of "k" and guanine and an oligonucleotide of "k" of thymine.

In addition, each primer of the primer sets (1)-(3) mentioned above may add the conventionally well-known arbitrary sequence to 5 'terminal, for example in order to raise reaction temperature of an amplification reaction.

The primer set for the obesity gene amplification of the present invention containing at least one of such primer sets (1) to (3) is preferably used for amplifying the obesity gene in a biological sample such as a whole blood sample. In particular, when using the primer set for the obesity gene amplification of the present invention together with a polymorphic detection probe as described later, it is preferable to make the addition ratio of the whole blood sample in the gene amplification reaction solution 0.1 to 0.5% by volume. Do. This point is mentioned later.

Reagents for amplifying obesity genes

Obesity gene amplification reagent of the present invention, as described above, a reagent for amplifying the obesity gene by the gene amplification method, at least one gene selected from the group consisting of the β2AR gene, β3AR gene and UCP1 gene And, characterized in that it comprises a set of primers for amplifying the obesity gene of the present invention. The reagent for amplifying obesity genes of the present invention is characterized by including the primer set of the present invention.

The reagent for amplifying obesity genes of the present invention may include a probe capable of hybridizing to a site to be detected of the obesity gene, for example, to detect an amplification product obtained by a gene amplification method using the primer set of the present invention. good. As described above, according to the primer set of the present invention, for example, depending on the kind of primer sets (1) to (3) included therein, each target region of three types of obesity genes is specific by gene amplification. Can be amplified. For this reason, by coexisting probes complementary (hybridizable) to the detection target sequence in the target region of the obesity gene, for example, the presence or absence of amplification, the genotype (polymorphism) of the detection target site, and the like will be described later. It is possible to detect by the method. Such a probe and its usage will be described in the following polymorphic analysis method. Moreover, it is preferable to use the reagent for amplifying obesity gene of this invention when amplifying the obesity gene in biological samples, such as whole blood. In particular, when the obesity gene amplification reagent of the present invention is used in combination with the above-described probe, it is preferable that the addition ratio of the whole blood sample in the reaction solution for gene amplification is 0.1 to 0.5% by volume. In addition, in this invention, a "detection target sequence" means the sequence containing the site | part (detection site | part) which a polymorphism generate | occur | produces.

The form of the obese gene amplification reagent of the present invention is not particularly limited, and may be, for example, a liquid reagent containing the primer set for obesity gene amplification of the present invention, or may be a dry reagent suspended in a solvent before use. In addition, the content of the primer set for obesity gene amplification is not particularly limited.

<Method of producing amplification product>

As described above, the method for producing an amplification product of the present invention is a method for producing an amplification product of an obesity gene by a gene amplification method, wherein the obesity gene is at least selected from the group consisting of β2AR gene, β3AR gene and UCP1 gene. One gene is characterized by including the following (I) process.

(I) At least one gene selected from the group consisting of the above β2AR gene, β3AR gene and UCP1 gene in the reaction solution, using the nucleic acid in the sample as a template and using the primer set for amplification of the obesity gene of the present invention. Process of amplifying

As described above, by performing the amplification reaction using the primer set of the present invention, the target region of the obesity gene can be amplified. Moreover, when the primer set for obesity gene amplification of this invention contains any two types of said primer sets (1)-(3), it contains each detection target site with respect to any two types of obesity genes. The target region can be amplified simultaneously in the same reaction solution. In addition, when the primer set for obesity gene amplification of the present invention includes all of the primer sets (1) to (3), the target region includes respective detection target sites for all of the three types of obesity genes. Can be amplified simultaneously in the same reaction solution. As described above, the target region to be amplified by the present invention is a region including a detection target site where the polymorphism of each target in each obesity gene occurs. Moreover, the manufacturing method of the amplification product of this invention is characterized by using the primer set of this invention, The kind, conditions, etc. of a gene amplification method are not restrict | limited at all.

The gene amplification method is not particularly limited as described above, and for example, a polymerase chain reaction (PCR) method, a nucleic acid sequence based amplification (NASBA) method, a transcription-mediated amplification (TMA) method, and a strand displacement amplification (SDA) method. The method etc. are mentioned, but PCR method is preferable. In addition, although this invention is demonstrated below using the PCR method as an example, it does not restrict to this.

As the sample to which the present invention is applied, for example, a nucleic acid as a template may be included, and the sample is not particularly limited. For example, the sample is preferably applied to a sample containing contaminants. Examples of the sample containing the contaminants include whole blood, intraoral cells (eg, oral mucosa), somatic cells such as nails and hair, germ cells, sputum, amniotic fluid, paraffin-embedded tissue, urine, and gastric juice (eg, stomach). Washing liquid), these suspensions, etc. are mentioned. According to the method for producing an amplification product using the primer set of the present invention, even a sample containing various contaminants (particularly, a biological sample such as whole blood or intraoral cells) is hardly affected by the three kinds of obesity genes. Each target region of can be specifically amplified. For this reason, according to this invention, even if it is a sample with many contaminants which were difficult in the conventional method, it can be used as it is, without performing pretreatment, such as purification, for example. Therefore, it can also be said that it is possible to prepare an amplification product more quickly than the conventional method from the viewpoint of sample pretreatment.

The addition ratio of the sample in the said reaction liquid is not specifically limited. As a specific example, when the sample is a biological sample (for example, a whole blood sample), the lower limit of the addition ratio in the reaction solution is preferably, for example, 0.01 vol% or more, more preferably 0.05 vol% or more, further Preferably it is 0.1 volume% or more. The upper limit of the addition ratio is not particularly limited, but is preferably, for example, 2 vol% or less, more preferably 1 vol% or less, and even more preferably 0.5 vol% or less.

In addition, in the case of the purpose of optical detection as described later, in particular, when performing optical detection using a labeled probe, the addition ratio of a biological sample such as a whole blood sample is preferably set to 0.1 to 0.5% by volume. . In the PCR reaction, heat treatment is usually performed for DNA denaturation (dissociation into single-stranded DNA). However, the heat treatment may denature sugars, proteins, and the like contained in the sample, resulting in insoluble precipitates and turbidity. have. For this reason, when the presence or absence of an amplification product and the genotype (polymorphism) of a detection target site are confirmed by an optical method, generation of such a precipitate and turbidity may affect measurement accuracy. However, if the addition ratio of the whole blood sample in the reaction solution is set in the above-mentioned range, the mechanism is unclear. However, since the influence due to the generation of precipitates due to denaturation and the like can be sufficiently prevented, the measurement by the optical method is performed. The precision can be improved. In addition, since inhibition of PCR by the contaminants in the whole blood sample is also sufficiently suppressed, the amplification efficiency can be further improved. Therefore, in addition to the use of the primer set of the present invention, the need for pretreatment of the sample can be further eliminated by setting the addition ratio of samples such as whole blood samples to the above-mentioned range.

In addition, the ratio of the whole blood sample in the said reaction liquid can also be represented by the weight ratio of hemoglobin (henceforth "Hb") instead of the volume ratio (for example, 0.1-0.5 volume%) mentioned above. In this case, the ratio of the whole blood sample in the reaction solution is preferably in the range of 0.565 to 113 g / L, more preferably in the range of 2.825 to 56.5 g / L, more preferably in terms of Hb amount. Preferably in the range of 5.65 to 28.25 μg / L. In addition, the addition ratio of the whole blood sample in the said reaction may satisfy both the said volume ratio and Hb weight ratio, for example, and may satisfy either.

The whole blood may be, for example, any of hemolyzed whole blood, whole blood of cosmetic blood, anticoagulant whole blood, whole blood containing a coagulation fraction, or the like.

In the present invention, the target nucleic acid contained in the sample is, for example, DNA. The DNA may be, for example, DNA originally contained in a sample such as a biological sample, or may be amplified product DNA amplified by a gene amplification method. In the latter case, cDNA produced by reverse transcription reaction (for example, reverse transcription PCR (RT-PCR)) from RNA (total RNA, mRNA, etc.) originally included in the sample may be mentioned.

In the method for producing an amplification product of the present invention, it is preferable to further add albumin to the reaction solution prior to the start of the gene amplification reaction. Thus, by adding albumin, the influence by the generation | occurrence | production of a deposit and turbidity as mentioned above can be further reduced, and amplification efficiency can also be improved further. Specifically, it is preferable to add albumin before the amplification reaction in the step (I) or the dissociation step into single-stranded DNA.

The addition ratio of albumin in the said reaction liquid is the range of 0.01-2 weight%, for example, Preferably it is 0.1-1 weight%, More preferably, it is 0.2-0.8 weight%. The albumin is not particularly limited, and examples thereof include bovine serum albumin (BSA), human serum albumin, rat serum albumin, and horse serum albumin. These may be any one type or a combination of two or more types.

Next, in the method for producing the amplification product of the present invention, a DNA for a whole blood sample is used as a target nucleic acid, and the primer set for the obesity gene amplification of the present invention comprising the primer sets (1) to (3) is used. By PCR, an example in which each amplified product of the β2AR gene, the β3AR gene, and the UCP1 gene is simultaneously produced in the same reaction solution will be described. Moreover, this invention is characterized by using the primer set of this invention, and other structure and conditions are not restrict | limited at all.

First, a PCR reaction solution is prepared. Although the addition ratio of the primer set of this invention is not restrict | limited, It is preferable to add F primer of primer sets (1)-(3) so that it may become 0.1-2 micromol / L, respectively, More preferably, it is 0.25- 1.5 μmol / L, particularly preferably 0.5 to 1 μmol / L. Moreover, it is preferable to add R primer of primer sets (1)-(3) so that it may become 0.1-2 micromol / L, respectively, More preferably, it is 0.25-1.5 micromol / L, Especially preferably, it is 0.5-1 μmol / L. The addition ratio (F: R, molar ratio) of the F primer and the R primer in each primer set is not particularly limited. For example, 1: 0.25 to 1: 4 are preferable, and more preferably 1: 0.5 to 1: 2.

Although the ratio of the whole blood sample in a reaction liquid is not specifically limited, The above-mentioned range is preferable. The whole blood sample may be added to the reaction solution as it is, or may be added to the reaction solution after dilution with a solvent such as water or a buffer solution in advance. When the whole blood sample is diluted in advance, the dilution rate is not particularly limited. For example, the final whole blood addition ratio in the reaction solution may be set to fall within the above range, but is, for example, 100 to 2000 times, preferably 200 to 1000. It is a ship.

The other composition component in the said reaction liquid is not specifically limited, A conventionally well-known component is mentioned, The ratio is not specifically limited, either. As said composition component, a DNA polymerase, a nucleotide (nucleoside triphosphate (dNTP)), and a solvent are mentioned, for example. In addition, as described above, the reaction solution preferably further contains albumin. In addition, in the said reaction liquid, the addition order of each composition component is not restrict | limited at all.

There is no restriction | limiting in particular as said DNA polymerase, For example, the polymerase derived from a conventionally well-known heat resistant bacterium can be used. Specific examples include DNA polymerases derived from Thermus aquaticus (US Pat. No. 4,889,818 and US Pat. No. 5,079,352) (trade name Taq polymerase), DNA polyps derived from Thermus thermophilus . Merase (WO 91/09950) (rTth DNA polymerase), DNA polymerase (WO 92/9688) derived from Pyrococcus furiosus (Pfu DNA polymerase: produced by Stratagenes), Thermococcus littorally ( DNA polymerase derived from Thermococcus litoralis ) (EP-A 455 430) (trademark Vent: manufactured by Biolab New England) and the like are commercially available, and among them, heat-resistant DNA poly derived from Thermus aquaticus Merases are preferred.

The addition ratio of the DNA polymerase in the reaction solution is not particularly limited, but is, for example, 1 to 100 U / mL, preferably 5 to 50 U / mL, and more preferably 20 to 30 U / mL. . In addition, the active unit (U) of the DNA polymerase generally uses activated salmon sperm DNA as a template primer, and acid-insoluble total nucleotides of 10 nmol in 30 minutes at 74 ° C in the reaction solution for measuring activity. The activity taken up in the precipitate is 1U. The composition of the reaction solution for measuring activity is, for example, 25 mM TAPS buffer (pH9.3, 25 ° C.), 50 mM KCl, 2 mM MgCl ₂ , 1 mM mercaptoethanol, 200 μM dATP, 200 μM dGTP, 200 μM dTTP, 100 μM “α- ³² P” dCTP, 0.25 mg / mL activated salmon sperm DNA.

As said nucleoside triphosphate, dNTP (dATP, dCTP, dTTP) is mentioned normally. Although the addition rate of dNTP in the said reaction liquid is not specifically limited, For example, it is 0.01-1 mmol / L, Preferably it is 0.05-0.5 mmol / L, More preferably, it is 0.1-0.3 mmol / L.

Examples of the solvent include buffers such as Tris-HCl, Tricine, MES, MOPS, HEPES, and CAPS. Commercially available PCR buffers and buffers of commercial PCR kits can be used.

In addition, the PCR reaction solution may further contain heparin, betaine, KCl, MgCl ₂ , MgSO ₄ , glycerol and the like, and their addition ratio may be set within a range that does not inhibit the PCR reaction, for example.

The total volume of the reaction solution is not particularly limited, and can be appropriately determined depending on, for example, a device (thermal cycler) used, but is usually 1 to 500 µL, preferably 10 to 100 µL. .

Next, PCR is performed. The cycle conditions of PCR are not particularly limited, but, for example, (1) dissociation of whole-blood-derived double-stranded DNA into single-stranded DNA, (2) annealing of the primer, and (3) elongation (polymerase reaction) of the primer are as follows. Same as The number of cycles is not particularly limited either, but three cycles of the following (1) to (3) are used as one cycle, for example, 30 cycles or more are preferable. Although an upper limit in particular is not restrict | limited, For example, it is 100 cycles or less in total, Preferably it is 70 cycles or less, More preferably, it is 50 cycles or less. What is necessary is just to control the temperature change of each step automatically using a thermal cycler etc., for example. When the primer set of the present invention is used, the amplification efficiency is excellent as described above, and according to the conventional method, it requires about 3 hours in 50 cycles. According to the present invention, it is about 1 hour (preferably It is also possible to complete 50 cycles in less than 1 hour).

As described above, amplification products of the respective target regions of the β2AR gene, the β3AR gene, and the UCP1 gene can be produced simultaneously in the same reaction solution. In addition, when producing an amplification product complementary to each target region of any one kind or any two kinds of obesity genes of three kinds of obesity genes, for example, it corresponds to the target region in primer sets (1) to (3). What is necessary is just to use the primer set for obesity gene amplification of this invention containing any 1 type or 2 types of primer sets.

The manufacturing method of the amplification product of this invention may also include the process of detecting the amplification product obtained by the amplification reaction mentioned above. Thereby, the presence or absence of an amplification product and genotype in the target region of the said obesity gene can also be detected. The presence or absence of an amplification product can be confirmed by a conventionally well-known method. Specifically, in the step (I), for example, a hybridizable probe (for example, a fluorescent labeling probe) is added to the reaction solution to a detection target site of the β2AR gene, the β3AR gene, or the UCP1 gene. Moreover, as (II) process, it can confirm by measuring the fluorescence intensity of the fluorescent label in the said probe about the said reaction liquid. In the case of two or three target regions to be amplified, for example, in the step (I), the hive is added to the reaction solution and to each detection target site of any one of β2AR gene, β3AR gene, and UCP1 gene. Two or three kinds of redistributable probes (e.g., fluorescent labeling probes) are added, and (II) step is confirmed by measuring the fluorescence intensity of each fluorescent label in each probe with respect to the reaction solution. Can be. In addition, detection of the polymorphism in an obesity gene is demonstrated below as one aspect of this invention.

Polymorphic interpretation of obesity genes

The polymorphism analysis method of the present invention is a method for analyzing a polymorphism of a detection target site in an obesity gene, wherein the obesity gene is at least one gene selected from the group consisting of β2AR gene, β3AR gene, and UCP1 gene. (Iii) to (iii).

(Iii) amplifying a region in the reaction solution containing a region to be detected in at least one gene selected from the group consisting of the β2AR gene, the β3AR gene, and the UCP1 gene by the method for producing an amplification product of the present invention

(Ii) a reaction solution containing a probe capable of hybridizing to the amplification product in step (iii) and at least one gene selected from the group consisting of the β2AR gene, the β3AR gene, and the UCP1 gene. Process to prepare

(Iii) changing a temperature of the reaction solution and measuring a signal value indicating a state of melting of the hybridization product between the amplification product and the probe

(Iii) determining the polymorphism of the detection target site in at least one gene selected from the group consisting of the β2AR gene, the β3AR gene and the UCP1 gene from the change in the signal value according to the temperature change.

As described above, the amplification product is prepared using the primer set of the present invention, thereby amplifying the target region including the polymorphism detection site in the obesity gene of at least one of the β2AR gene, the β3AR gene, and the UCP1 gene. Thus, the polymorphism of the detection target site in the target region can be analyzed.

The probe in the step (iii) is not particularly limited, and probes that hybridize to the detection target site of the β2AR gene (hereinafter also referred to as the "β2AR gene probe") are hybridized to the detection target site of the β3AR gene. It is preferable that these probes (hereinafter also referred to as "β3AR gene probe") and probes which hybridize to the detection target site of the UCP1 gene (hereinafter also referred to as "UCP1 gene probe"). It is preferable that these probes are probes which are complementary to the detection object sequence containing the said detection object sequence. Any one type of these probes may be used, or any two or all three types thereof may be used. For example, the probes may be determined according to the type of the target region amplified by the primer set for obesity gene amplification of the present invention. When two or three types of probes are used, for example, the same reaction solution can be used to analyze any two detection sites or polymorphisms of all three detection sites.

The probe for the obese gene is not particularly limited, and can be set by a conventionally known method, and is designed based on the sequence of the sense chain of each obesity gene, for example, as a detection target sequence including a polymorphism detection site. You may design based on the sequence of an antisense chain. In addition, the base of the detection target site of a polymorph can be suitably determined according to the kind of each polymorph. That is, in the case of the β2AR gene, since the polymorphisms of "A" and "G" are known in the 4265th base in SEQ ID NO: 1, for example, the detection target sequence in which the 4265th is A and the 4265th G are detected. And a probe (a probe for detecting a sense strand) complementary to any one of the target sequences, or a probe (a probe for detecting an antisense strand) complementary to the sequence of the antisense strand. In the case of the β3AR gene, since the polymorphisms of "T" and "C" are known in the 4134th base in SEQ ID NO: 2, for example, the detection target sequence in which the 4134th is T and the 4265th is C are detected. And a probe (a probe for detecting a sense strand) complementary to any one of the target sequences, or a probe (a probe for detecting an antisense strand) complementary to the sequence of the antisense strand. In the case of the UCP1 gene, since the polymorphisms of "A" and "G" are known in the 292th base in SEQ ID NO: 3, for example, the detection target sequence where the 4134th is A and the 4265th G are detected. And a probe (a probe for detecting a sense strand) complementary to any one of the target sequences, or a probe (a probe for detecting an antisense strand) complementary to the sequence of the antisense strand. In this way, even if the probe is designed by setting the base of the detection target site where the polymorphism occurs to any one of the bases described above, a method as described later indicates a certain polymorphism at the detection target site of each obesity gene. It is possible to judge whether

Each probe may be added to the amplification reaction solution after the step (i), that is, after performing an amplification reaction on the target region of the obesity gene, but in that it can be easily and quickly analyzed. It is preferable to add to reaction liquid beforehand before the amplification reaction of the said (iii) process.

Although the addition ratio of the probe in the said reaction liquid is not specifically limited, For example, it is preferable to add each probe so that it may become the range of 10-400 nmol, More preferably, it is 20-200 nmol. When a fluorescent dye is used as a label for the probe, for example, in order to adjust the fluorescence intensity to be detected, an unlabeled probe having the same sequence as the labeled probe may be used in combination, and the unlabeled probe may be used at its 3 'end. Phosphoric acid may be added. In this case, the molar ratio of the labeled probe and the unlabeled probe is preferably 1:10 to 10: 1, for example. The length of the probe is not particularly limited and is, for example, 5 to 50 mer, and preferably 10 to 30 mer.

The Tm value will be described. When the solution containing double-stranded DNA is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both chains in double-stranded DNA are released by heating to dissociate into single-stranded DNA (fusion of DNA). And when all the double-stranded DNA dissociates and becomes single-stranded DNA, the absorbance shows about 1.5 times the absorbance at the start of heating (absorbance only of the double-stranded DNA), and it can be judged that fusion is completed by this. Based on this phenomenon, the melting temperature Tm is generally defined as the temperature when the absorbance reaches 50% of the total increase in absorbance.

In the step (iii), the measurement of the signal representing the melting state of the hybridization product between the amplification product and the probe may be the above-described absorbance measurement at 260 nm, or may be a signal measurement of a labeling substance. Specifically, it is preferable to perform signal measurement of the labeling substance using a labeling probe labeled with a labeling substance as the probe. As said labeling probe, the labeling probe which shows a signal by itself and does not show a signal by hybrid formation, or the labeling probe which does not show a signal by itself and shows a signal by hybrid formation is mentioned, for example. If the probe is the same as the former, the signal is not displayed when a hybrid (double-stranded DNA) is formed with the sequence to be detected, and the signal is displayed when the probe is favored by heating. In the latter probe, a signal is obtained by forming a hybrid (double-stranded DNA) with the sequence to be detected, and the signal decreases (dissipates) when the probe is favored by heating. Therefore, by detecting the signal according to the label under conditions specific to the signal (absorption wavelength or the like), the advancing of the melting and determination of the Tm value and the like can be performed similarly to the absorbance measurement at 260 nm.

In the present invention, each polymorphism can be confirmed for the amplification products of two or three kinds of obesity genes amplified by the same reaction solution. For this reason, when using two or three types of probes, it is preferable that they are labeled with different labels (for example, fluorescent labels) detected under different conditions. Thus, by using different labels, even if it is the same reaction liquid, it becomes possible to analyze each amplification product separately by changing detection conditions.

As a specific example of the labeling substance in the said labeling probe, fluorescent dye (fluorophore) is mentioned, for example. As a specific example of the labeling probe, for example, a probe that is labeled with a fluorescent dye and exhibits fluorescence alone and decreases fluorescence by hybrid formation (for example, quenching) is preferable. A probe using such a fluorescence quenching phenomenon is generally called a fluorescence quenching probe. Especially, as said probe, it is preferable that the 3 'end or 5' end of an oligonucleotide is labeled with the fluorescent dye, and it is preferable that the base of the said terminal to be labeled is C. In this case, in the detection target sequence hybridized by the labeling probe, the labeling probe is G such that a base paired with the terminal base C of the labeling probe or a base 1 to 3 bases away from the pairing base becomes G. It is preferable to design the base sequence of. Such probes are generally called guanine quenching probes and are known as so-called QProbe®. When such a guanine quenching probe hybridizes to the detection target sequence, C at the terminal labeled with the fluorescent dye approaches G in the detection target DNA, resulting in weak emission of the fluorescent dye (reduction in fluorescence intensity). Indicates a phenomenon. By using such a probe, hybridization and dissociation can be easily confirmed by the change of a signal.

Although it does not restrict | limit especially as said fluorescent dye, For example, a fluorescein, a phosphor, rhodamine, a polymethine pigment derivative, etc. are mentioned, As a commercial fluorescent dye, BODIPY FL (trademark, a molecular probe company make) is mentioned, for example. FluorePrime (trade name, manufactured by Amersham Pharmacia), Fluoredite (trade name, manufactured by Millipore), FAM (manufactured by ABI), Cy3 and Cy5 (manufactured by Amersham Pharmacia), TAMRA (molecular probe company) Production). The combination of fluorescent dyes used for the three types of probes may be detected under different conditions, for example, and is not particularly limited. Examples thereof include Pacific Blue (detection wavelength 450 to 480 nm) and TAMRA (detection wavelength 585 to 700 nm). ) And BODIPY FL (detection wavelength 515-555 nm), etc. are mentioned.

Below, the specific example of the sequence of the probe for detecting each polymorphism of the above-mentioned (beta) 2AR gene, (beta) 3AR gene, and UCP1 gene is shown. In addition, this invention is not limited to this. The following probe (P1) is an example of a β2AR gene probe, (1-1) is a probe for detecting a sense chain, and (1-2) is a probe for detecting an antisense chain. The following probe (2) is an example of a β3AR gene probe, (2-1) is a probe for detecting an antisense chain, and (2-2) is a probe for detecting a sense chain. The following probe (3) is an example of a probe for a UCP1 gene, and is a probe for detecting an antisense strand. In addition, each probe P1-(P3) may be one type, respectively, and may use two or more types together, respectively, as mentioned later.

Probe  ( P1 )

(1-1) At least one oligonucleotide complementary to the region from the 21st to 26th base in the 3 'direction with the 4254th thymine base (T) in SEQ ID NO: 1 as the first base, wherein the thymine Oligonucleotide having 3 'end of adenine base (A) complementary to base

(1-2) At least one oligonucleotide having the same sequence as the region from the 15th to the 19th base in the 3 'direction using the 4259th cytosine base (C) in SEQ ID NO: 1 as the first base; Oligonucleotide with 5 'terminal of cytosine base

Probe  ( P2 )

(2-1) At least one oligonucleotide having the same sequence as the region from the 17th to the 28th base in the 5 'direction with the 4140 th cytosine base (C) in SEQ ID NO: 2 as the first base, as described above. Oligonucleotides with 3 'end of cytosine base

(2-2) At least one oligonucleotide complementary to the region from the 12th to the 17th base in the 5 'direction with the 4144th guanine base (G) in SEQ ID NO: 2 as the first base, wherein the guanine Oligonucleotide having 5 'end of cytosine base (C) complementary to base

Probe  ( P3 )

At least one oligonucleotide having the same sequence as the region from the 17th to the 31th base in the 3 'direction with the 280th cytosine base (C) in SEQ ID NO: 3 as the first base, wherein the cytosine base is 5'. Oligonucleotide terminated

In the probe (1-1), the base complementary to position 4265 of SEQ ID NO: 1 is represented by y, and y is T or C. In the probe (1-2), the base corresponding to 4265th SEQ ID NO: 1 is represented by r, and r is A or G. In the probe (2-1), the base corresponding to 4134th of SEQ ID NO: 2 is represented by y, and y is T or C. In the probe (2-2), the base complementary to sequence 4134 of SEQ ID NO: 2 is represented by r, wherein r is A or G. In the probe (3), the base corresponding to 292st of SEQ ID NO: 3 is represented by r, and r is A or G.

In addition, specific examples of the probe (1), the probe (2) and the probe (3) are shown in the following table. "Tm (degreeC)" in the following table | surface is Tm (degreeC) when the sequence completely complementary with the sequence of the following table | surface is a parameter by MELTCALC software (http://www.meltcalc.com/). It is the value computed by making oligonucleotide concentration of 0.2 micrometer, and sodium equivalent (Naeq.) 50 mM.

In the above table, oligonucleotides of SEQ ID NOs: 69 to 74 are specific examples of the probe (1-1), and "y" in each nucleotide sequence may be any of T and C, as shown below. good. In the above table, the probe (1-1) represented by SEQ ID NO: 69 to SEQ ID NO: 74 where "y" is C is complementary to the β2AR gene (sense chain) in which the 4265th base of SEQ ID NO: 1 is G. Perfect match), and the uppercase base C represents a base complementary to base 4, G265 of SEQ ID NO: 1. In the said table | surface, as a specific example, the sequence of the probe whose "y" is "C" and the Tm value (degreeC) when the said probe and the sequence completely complementary to it are hybridized are shown.

5'-cgcatggcttcyattgggtgcca-3 '(SEQ ID NO: 72: y)

5'-cgcatggcttccattgggtgcca-3 '(SEQ ID NOs: 72, 101: y is C)

5'-cgcatggcttctattgggtgcca-3 '(SEQ ID NOs: 72, 102: y is T)

In the above table, oligonucleotides of SEQ ID NOs: 112 to 116 are specific examples of the probe (1-2), and "r" in each nucleotide sequence may be any of A and G as described above. . In the above table, the probe (1-2) represented by SEQ ID NO: 112 to SEQ ID NO: 116 in which "r" is G is a complementary chain (antisense chain) of the β2AR gene in which the 4265th base of SEQ ID NO: 1 is G. It is a probe complementary to (perfect match), and an uppercase base represents the 4265th base of SEQ ID NO: 1. In the said table | surface, as a specific example, the sequence of the probe whose "r" is "G" and the Tm value (degreeC) when the said probe and the sequence completely complementary to it are hybridized are shown.

In the above table, the probe (2-1) represented by SEQ ID NOs: 75 to 86 is composed of the same sequence as the region in which the 4134th in SEQ ID NO: 2 is C, and the base of the capital letter is represented by SEQ ID NO: 2. The 4134th base is shown. In the probe (2-1), the base of the capital letter may be substituted with y, and y may be any of C and T. In the above table, the probe (2-2) represented by SEQ ID NOs: 117 to 128 is composed of a sequence complementary to a region in which the 4134th in SEQ ID NO: 2 is C, and the base of the capital letter is SEQ ID NO: 2 The base complementary to the 4134th base of is shown. In addition, in the said probe (2-2), the base of the said capital letter can be substituted by r, and said r may be any of G and A. In the above table, the probes (3) represented by SEQ ID NOs: 87 to 100 and 139 consist of the same sequence as the region in which the 292th in SEQ ID NO: 3 is G, and the base of the uppercase letter is the 292nd in SEQ ID NO: 3 Represents the base. In addition, in the said probe (3), the base of a capital letter can be substituted by r, and said r may be any of G and A. As a specific example of the probe in the present invention, for example, as described above, the complementary chain of the oligonucleotide shown in the above table may be used.

The said probe is an example, and this invention is not limited to this at all. As the β2AR gene probe, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 72 is preferable among the probes (1-1), and "y" may be any of T and C as described above. Among the probes (1-2), oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 114 are preferable, and "r" may be any of A and G. In addition, as the β2AR gene probe, it is preferable to use a so-called wild type detection probe and a variant detection probe together. Here, the wild-type detection probe is, for example, a probe for detecting a detection target sequence (sense chain) or its complementary strand (antisense chain) in which the 4265th base of SEQ ID NO: 1 is A (or G). The probing probe is a probe for detecting a detection target sequence (sense chain) or its complementary strand (antisense chain) in which the 4265th base of SEQ ID NO: 1 is G (or A). In the present invention, among the probes (1-1), for example, at least one oligonucleotide (mutation detection probe) consisting of the nucleotide sequences of SEQ ID NOs: 69 to 74 in which "y" is "C", and "y" It is preferable to use together at least one oligonucleotide (wild type detection probe) which consists of the base sequences of SEQ ID NO: 69-74 whose "T" is, More preferably, to SEQ ID 72 which "y" is "C" It is combined use with the oligonucleotide (mutant detection probe) which consists of, and the oligonucleotide (wild-type detection probe) which consists of the nucleotide sequence of SEQ ID NO: 72 whose "y" is "T".

The inventors separately conducted a study on the analysis of polymorphisms, and as a result, when the β2AR gene polymorphism (polymorphism of the region encoding amino acid position 16 of β2AR: polymorphism of the 4265th base of SEQ ID NO: 1) was analyzed, According to the used Tm analysis, the knowledge that signal detection was difficult was acquired. Thus, the present inventors conducted further studies, and as a result, if the above-described probe (1), in particular, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 72 (that is, the oligonucleotide of SEQ ID NO: 101 and the oligonucleotide of SEQ ID NO: 102) As described later, it was found that the signal in the case of matching the amplification product of the β2AR gene and the signal in the case of mismatching can be sufficiently distinguished and detected. The probe represented by SEQ ID NO: 101 is a probe that perfectly matches the detection target sequence of the β2AR gene (sense chain) having the 4265th base of SEQ ID NO: G, and the probe represented by SEQ ID NO: 102 is the 4265th SEQ ID NO: 1 It is a probe which matches the detection target sequence of the (beta) 2AR gene (sense chain) whose base of A is perfect. In addition, the present invention is characterized by amplifying each target region of the three types of genes simultaneously and specifically in the same reaction solution by using the primer set for the obesity gene amplification as described above. The sequence of the probe used for analysis of the obtained amplification product is not specifically limited.

As the probe for the β3AR gene, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 83 is preferable among the probes (2-1). As the probe (2-2), it is preferable to use a so-called wild type detection probe and a variant detection probe together. Here, the wild-type detection probe is, for example, a probe for detecting a detection target sequence (sense chain) or its complementary strand (antisense chain) in which the 4134th base of SEQ ID NO: 2 is T, and the variant detection probe is It is a probe for detecting the detection object sequence (sense chain) whose 4134th base of sequence number 2 is C, or its complement chain (antisense chain). In the present invention, for example, at least one oligonucleotide (mutation detection probe) consisting of the nucleotide sequences of SEQ ID NOs: 117-122, and at least one oligonucleotide consisting of the nucleotide sequences of SEQ ID NOs: 123-128 (for wild-type detection) It is preferable to use together the probe), More preferably, use the oligonucleotide (mutation detection probe) which consists of a base sequence of SEQ ID NO: 120, and the oligonucleotide (wild type detection probe) which consists of a base sequence of SEQ ID NO: 127 together It is. Thus, when using a wild type detection probe and a variant detection probe together, it is preferable to set, for example, shifting the Tm value of the perfect match of each probe.

As the probe for the UCP1 gene, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 95 or SEQ ID NO: 139 is preferable among the probes (3).

And when using two or more types of these probes, it is preferable to label with each fluorescent dye (fluorescent dye detected by different wavelength) as above-mentioned. For example, when the probes shown in the above table are used as quenching probes, the probes 1-1 and 2-1 are each labeled with the fluorescent dye (for example, Pacific Blue, BODIPY FL, etc.) as described above. It is preferable to label, and it is preferable that the probe (1-2), the probe (2-2), and the probe (3) label the cytosine at the 5 'end with the fluorescent dye (for example, TAMRA or the like) as described above. . In addition, although the 3 'end of the probe (1-1) shown in the said table is adenine (A), since the quenching can be confirmed by hybridizing adjacent cytosine (C) with guanine (G), as a guanine quenching probe Can be used. In addition, the probe in which the fluorescent dye is labeled at the 5 'end may be further added with a phosphate group at the 3' end, for example, in order to prevent the probe itself from elongating. In addition, when using a wild-type detection probe and a mutant detection probe as mentioned above, each fluorescent dye may be same or different.

Next, as an example, the detection method of the present invention, using the following probe, polymorphism of the β2AR gene (polymorph of the 4265th base in SEQ ID NO: 1), and polymorphism of the β3AR gene (SEQ ID NO: 2). A method for detecting the 4134th base polymorphism) and the UCP1 gene polymorphism (292th base polymorphism in SEQ ID NO: 3) will be described. In addition, this invention is not limited to this.

(Probe)

Probe for β2AR Gene

5'-cgcatggcttcCattgggtgcca- (Pacific Blue) -3 '(SEQ ID NO: 72), or

5 '-(Pacific Blue) -cccaatGgaagccatgc-P-3' (SEQ ID NO: 114)

Probe for β3AR Gene

5'-cgtggccatcgccCggactc- (BODIPY FL) -3 '(SEQ ID NO: 83), or;

5 '-(BODIPY FL) -ctcggagtccGggc-P-3' (SEQ ID NO: 120), and

5 '-(BODIPY FL) -ctcggagtccAggc-P-3' (SEQ ID NO: 127)

UCP1 Gene Probe

5 '-(TAMRA) -cacagtttgatcGagtgcatttg-3' (SEQ ID NO: 95), or

5 '-(TAMRA) -cacagtttgatcGagtg-3' (SEQ ID NO: 139)

First, PCR is performed as described above using the reaction solution to which the above three types of labeling probes are added, and simultaneously amplify each target region of the β2AR gene, the β3AR gene, and the UCP1 gene in the same reaction solution.

Next, dissociation of the obtained amplification product and hybridization of the single-stranded DNA obtained by dissociation with the labeling probe are performed. This can be done, for example, by changing the temperature of the reaction solution.

The heating temperature in the dissociation step is not particularly limited as long as it is a temperature at which the amplification product can dissociate. For example, the heating temperature is 85 to 95 ° C. Although heating time in particular is not restrict | limited, either, Usually, they are 1 second-10 minutes, Preferably they are 1 second-5 minutes.

Hybridization of the dissociated single-stranded DNA with the labeling probe can be performed, for example, by lowering the heating temperature in the dissociation step after the dissociation step. As temperature conditions, it is 40-50 degreeC, for example.

And the temperature of the said reaction liquid is changed and the signal value which shows the melting state of the hybridization body of the said amplification product and the said labeling probe is measured. Specifically, for example, the reaction solution (hybrid formed body of the single-stranded DNA and the labeled probe) is heated, and the change in signal value accompanying the temperature rise is measured. As described above, for example, when a guanine quenching probe is used, fluorescence is reduced (or quenched) in the hybridized state with single-stranded DNA, and fluoresces in the dissociated state. Therefore, for example, what is necessary is just to gradually heat the hybrid formation in which fluorescence is decreasing (or quenching), and to measure the increase in fluorescence intensity accompanying a temperature rise.

Although the temperature range at the time of measuring the fluctuation | variation of fluorescence intensity | strength is not restrict | limited, For example, start temperature is room temperature-85 degreeC, Preferably it is 25-70 degreeC, and end temperature is 40-105 degreeC, for example. The rate of increase in temperature is not particularly limited, but is, for example, 0.1 to 20 ° C / sec, preferably 0.3 to 5 ° C / sec.

Next, the Tm value is determined by analyzing the fluctuation of the signal. Specifically, the fluorescence intensity change amount (-d fluorescence intensity increase amount / dt) per unit time at each temperature is calculated from the obtained fluorescence intensity, and the temperature showing the lowest value can be determined as the Tm value. Further, the point where the fluorescence intensity increase amount (fluorescence intensity increase amount / t) per unit time is the highest can be determined as the Tm value. As a labeling probe, when the probe which does not display a signal alone but shows a signal by hybrid formation is used instead of a quenching probe, the amount of decrease in fluorescence intensity may be measured on the contrary.

In the present invention, in order to detect each polymorphism of three genes, each Tm value is determined under conditions according to each label of the three types of probes. Pacific Blue of the β2AR gene probe is, for example, a detection wavelength of 450 to 480 nm, BODIPY FL of the β3AR gene probe is, for example, a detection wavelength of 515 to 555 nm, and a TAMRA of the UCP1 gene probe is, for example, a detection wavelength of 585 to Detection at 700 nm.

Then, from these Tm values, the genotype at the detection target site of each gene is determined. In the Tm analysis, a hybrid (match) that is completely complementary results in a higher Tm value indicating dissociation than a hybrid (mismatch) having a single base. Therefore, by determining the Tm values of the hybrids that are completely complementary to the probes and the Tm values of the hybrids having different monobases, the amplification products can be determined to match the probes or mismatches in advance. For example, assuming that the base of the target site in the detection target sequence is a variant (for example, the 4265th base in SEQ ID NO: 1 is G), and a probe complementary to the detection target sequence containing the same is used, formation is performed. If the Tm value of one hybrid is equal to the Tm value of a hybrid that is completely complementary, it can be determined that the polymorphism of the amplification product is variant. When the Tm value of the formed hybrid is the same as the Tm value of another hybrid of one base (a lower value than the Tm value of the completely complementary hybrid), the polymorphism of the amplification product is the wild type (4265th in SEQ ID NO: 1). It can be determined that the base is A). In addition, when both Tm values are detected, it can be determined that it is a hetero conjugate. In this way, the polymorphisms of the β2AR gene, the β3AR gene, and the UCP1 gene can be determined from the Tm values for the respective labeled probes. In addition, when confirming a Tm value by detection of fluorescence intensity, as mentioned above, using a non-labeled probe which is the same oligonucleotide sequence together as a probe so that a signal of a match and a signal of a mismatch can fully be distinguished. desirable. Among the β2AR gene probes, the β3AR gene probes, and the UCP1 gene probes, the addition of the unlabeled probes is not particularly limited. For example, it is preferable to add the labeled and unlabeled probes to the β3AR gene probes. .

In addition, in the present invention, as described above, instead of the method of raising the temperature of the reaction solution containing the probe (heating the hybrid forming body) and measuring the signal fluctuation accompanying the temperature rise, for example, a hybrid The signal fluctuation at the time of formation may be measured. That is, when dropping the temperature of the reaction liquid containing the probe to form a hybrid body, the signal fluctuation accompanying the temperature drop may be measured.

 As a specific example, when a labeling probe (eg, guanine quenching probe) using a signal alone and not displaying a signal by hybrid formation is used, fluorescence occurs in the state in which the single-stranded DNA and the probe dissociate. When a hybrid is formed by drop, the fluorescence is reduced (or quenched). Therefore, for example, the temperature of the reaction solution may be gradually lowered, and the decrease in fluorescence intensity accompanying the temperature drop may be measured. On the other hand, when a labeling probe that does not display a signal alone and exhibits a signal by hybrid formation is used, the single-stranded DNA and the probe do not fluoresce in the dissociated state, but when hybridization is formed by a drop in temperature, It will fluoresce. Therefore, for example, the temperature of the reaction solution is gradually lowered, and the increase in fluorescence intensity accompanying the temperature drop may be measured.

In addition, when analyzing the polymorphism of any one type or the polymorphism of any two types of genes among three types of obesity genes (β2AR gene, β3AR gene and UCP1 gene), for example, primer sets (1) to (3 ), Any one kind of hybridization to the target detection site using the primer set for the obesity gene amplification of the present invention, including any one or two kinds of primer sets corresponding to the target region What is necessary is just to use a probe or two types of probes.

<β2AR Gene Analysis Probe>

The obesity analysis probe of the present invention is a probe for use in polymorphism analysis of an obesity gene, wherein the obesity gene is a β2AR gene and is composed of an oligonucleotide shown below (P1).

(P1)

Oligonucleotide of at least one of following (1-1) and following (1-2)

(1-1) At least one oligonucleotide complementary to the region from the 21st to 26th base in the 3 'direction with the 4254th thymine base (T) in SEQ ID NO: 1 as the first base, wherein the thymine Oligonucleotide having 3 'end of adenine base (A) complementary to base

(1-2) At least one oligonucleotide having the same sequence as the region from the 15th to the 19th base in the 3 'direction using the 4259th cytosine base (C) in SEQ ID NO: 1 as the first base; Oligonucleotide with 5 'terminal of cytosine base

The probe consisting of the oligonucleotide shown in the above (P1) is the probe for the β2AR gene, as described above.

According to the probe for β2AR gene of the present invention, a detection target sequence including a region encoding the β2AR amino acid position 16 in the β2AR gene can be detected. Specifically, by using the probe of the present invention, for example, it is possible to perform the melting temperature analysis of the hybrid body of the probe of the present invention and the detection target sequence with excellent reliability, and by the melting temperature analysis, Analysis of the presence or absence of the detection target sequence in the sample and determination of the polymorphism (polymorphism of the 4265th base of SEQ ID NO: 1) of the detection target site in the detection target sequence can also be performed with excellent reliability. Therefore, the β2AR gene analysis probe of the present invention can be used for the polymorphic analysis of the β2AR gene by analysis of the β2AR gene, specifically, for example, analysis of melting temperature and analysis of melting temperature, as described below. Moreover, it can also be used for the polymorphic analysis method of the three types of obesity gene mentioned above. Therefore, the probe and analysis method for β2AR gene analysis of the present invention can be said to be a very useful tool / analysis method particularly in the medical field for treating or preventing obesity.

The β2AR gene probe may be, for example, an unlabeled probe to which a labeling substance is not added, but is preferably a labeling probe to which a labeling substance is added. A label site | part and a label substance are as above-mentioned.

<β2AR Gene Analysis Reagent>

The reagent for analyzing the obesity gene of the present invention is a reagent for use in analyzing the β2AR gene, and is characterized by including the β2AR gene analyzing probe of the present invention (hereinafter also referred to as "the reagent for analyzing the β2AR gene"). As described later, the reagent for analysis of the present invention can be used for analysis of the β2AR gene, specifically, for example, polymorphism analysis of the β2AR gene by analysis of melting temperature and analysis of melting temperature. Moreover, it can also be used for the polymorphic analysis method of the two or three types of obesity genes containing the (beta) 2AR gene. The β2AR gene analysis reagent of the present invention is characterized by including the β2AR gene analysis probe of the present invention, and the composition other than this is not limited at all.

It is preferable that the reagent for analyzing the β2AR gene of the present invention further includes a primer for amplifying the detection target sequence having the detection target site in the β2AR gene. As said primer, the primer set for the (beta) 2AR gene mentioned above is mentioned, for example. Thus, when the analysis reagent of the present invention including the primer set for the β2AR gene is used, for example, the amplification reaction of the β2AR gene can be performed, and the β2AR gene can be further analyzed using the amplification product obtained by the reaction. have. The region to be amplified by the β2AR gene primer may be a region including the detection target site in the β2AR gene. As a specific example, in the (beta) 2AR gene, the detection target sequence which has the said detection target site, ie, the sequence which hybridizes the probe of this invention, may be sufficient, and the area | region containing the said detection target sequence may be sufficient. Hereinafter, the area | region which amplifies by an amplification reaction is also called "a target area."

In addition, the reagent for analyzing the β2AR gene of the present invention is preferably used when analyzing the β2AR gene in a biological sample such as whole blood. In particular, the reagent for analyzing the β2AR gene of the present invention includes the primer set for the β2AR gene in addition to the β2AR gene analysis probe of the present invention, and when used for amplification of a detection target sequence, for example, in a reaction solution of an amplification reaction, It is preferable to make the addition ratio of the whole blood sample of 0.1 to 0.5 volume%. This point is as described above.

The form of the β2AR gene analysis reagent of the present invention is not particularly limited, and may be, for example, a liquid reagent containing the β2AR gene analysis probe of the present invention, or may be a dry reagent suspended in a solvent before use. The content of the β2AR gene analysis probe is not particularly limited.

The reagent for analyzing the β2AR gene of the present invention may further include a probe or primer for detecting another gene or another polymorphism, for example, in the same reaction solution. As described above, the polymorphism of the β3AR gene and the CYP2C9 gene, like the β2AR gene, is known to be an indicator of the treatment or prevention of obesity, and thus further includes a probe or primer set for detecting either or both. You may also Thus, for example, when amplifying target regions of a plurality of genes and detecting by a probe, the target region of the β2AR gene can be specifically amplified, so that the primer set for the β2AR gene as described above is contained. It is preferable to make it.

<β2AR Gene Analysis Method>

The obesity gene analysis method of this invention is an analysis method which analyzes the (beta) 2AR gene, It is characterized by including the following processes (a)-(c).

(a) preparing a reaction solution containing a detection target sequence having a polymorphism detection site in the β2AR gene and a probe for analyzing the β2AR gene of the present invention

(b) a step of changing a temperature of the reaction solution and measuring a signal value indicating a state of melting of the hybridization body between the detection target sequence and the β2AR gene analysis probe

(c) determining the melting temperature of the hybrid formed from the change in the signal value according to the temperature change

As described above, by using the β2AR gene analysis probe of the present invention, for example, it is possible to sufficiently detect a change in a signal value indicating the state of melting of the hybridization body between the detection target sequence and the β2AR gene analysis probe. For this reason, according to the β2AR gene analysis method of the present invention, it is possible to determine the melting temperature with respect to the sequence to be detected of the β2AR gene with high reliability. Thereby, for example, when nucleic acid amplification is performed on a region including the detection target site of the β2AR gene, it is also possible to judge whether the target region is amplified with high reliability by analyzing the melting temperature (Tm). Become. In addition, since the Tm value in case of perfect match between the probe of the present invention and the detection target sequence and the Tm value in case of mismatch show a sufficiently discernible difference, the β2AR gene of the test subject is the probe of the present invention. It is possible to fully distinguish between perfect match and miss match. Thus, if a perfect match and a miss match can be fully distinguished, the polymorphism of the (beta) 2AR gene of a test subject can be judged with more excellent reliability. Further, the β2AR gene analysis method of the present invention is characterized by using the β2AR gene analysis probe of the present invention. For example, the kind of the signal to be measured, the measuring method and the measurement conditions are not limited at all.

Since the β2AR gene analysis method of the present invention can determine the Tm value as described above, the step (d) may be further included.

(d) Step of determining polymorphism of detection target site of β2AR gene from determined melting temperature

The β2AR gene analysis method of the present invention may further include the following (x) step. According to this method, for example, it is possible to continuously amplify the β2AR gene, determine the Tm value, and analyze the polymorphism using the same reaction solution. In addition, as the following primer, the above-mentioned primer for β2AR gene can be used.

(x) A step of amplifying the detection target sequence in a reaction solution using a nucleic acid in the sample as a template and using a primer for amplifying the detection target sequence in the β2AR gene.

The gene amplification method in the step (x) is not particularly limited. For example, the above-described method can be employed, and the PCR method is preferable.

The β2AR gene analysis probe may be added to the reaction solution of the amplification reaction after the step (x), that is, after the amplification reaction of the β2AR gene, but can be easily and quickly analyzed. It is preferable to add to reaction liquid beforehand before the amplification reaction of the said (x) process. In this case, the reaction solution after the amplification reaction in the step (x) can be used as the reaction solution in the step (a). According to this method, it is possible to analyze the melting temperature by using the reaction solution subjected to the amplification reaction as it is.

Although the addition ratio of the probe in the said reaction liquid is not specifically limited, For example, it is preferable to add each probe so that it may become the range of 10-400 nmol, More preferably, it is 20-200 nmol. When a fluorescent dye is used as a label for the probe, for example, in order to adjust the fluorescence intensity to be detected, an unlabeled probe having the same sequence as the labeled probe may be used in combination, and the unlabeled probe may be used at its 3 'end. Phosphoric acid may be added. In this case, the molar ratio of the labeled probe and the unlabeled probe is preferably 1:10 to 10: 1, for example. The length of the probe is not particularly limited and is, for example, 5 to 50 mer, and preferably 10 to 30 mer.

In the step (b), the measurement of the signal value indicating the melting state of the hybridization body between the detection target sequence and the β2AR gene analysis probe is performed using the unlabeled probe, for example, at 260 nm. Although absorbance measurement may be performed, it is preferable to perform signal measurement of a labeling substance using a labeling probe. Specifically, as the β2AR gene analysis probe, it is preferable to perform signal measurement of the labeling substance using a labeling probe labeled with a labeling substance. As said labeling probe, the above-mentioned thing is mentioned, for example, It can measure similarly to the above-mentioned.

As the sample to which the present invention is applied, for example, a nucleic acid as a template may be included, and the sample is not particularly limited, and examples thereof include those mentioned above. In addition, the addition ratio of the sample in the said reaction liquid is as above-mentioned, for example.

Next, the β2AR gene analysis method of the present invention, as an example, amplifies the β2AR gene from a whole blood sample by PCR, and uses the β2AR gene analysis probe of the present invention to determine the polymorphism of the β2AR gene (SEQ ID NO: 1). The polymorphism of the 4265th base in the above) will be described. Moreover, unless otherwise indicated, it can carry out similarly to the polymorphic analysis method of the obesity gene of this invention mentioned above, In addition, it is not limited to this.

First, PCR reaction solution is prepared, PCR is performed, and the obtained amplification product is dissociated, and hybridization of the single-stranded DNA obtained by dissociation with the β2AR gene analysis probe is performed. Then, the reaction solution is heated (heating hybrid formation of the single-stranded DNA and the β2AR gene analysis probe), and the signal value of the reaction solution is measured. As described above, the measurement of the signal value can be appropriately determined according to the presence or absence of the label of the β2AR gene analysis probe to be used, the type of the labeling substance, and the like.

Next, the Tm value is determined by analyzing the fluctuation of the measured signal value. When the β2AR gene analysis probe is a fluorescently labeled probe, for example, the amount of fluorescence intensity change (-d fluorescence intensity change / dt or d fluorescence intensity change / dt) per unit time at each temperature is calculated from the obtained fluorescence intensity, The temperature representing the largest absolute value can be determined as the Tm value. For example, in the case of the guanine quenching probe, the temperature at which the -d fluorescence intensity increase / dt is the lowest is determined as the Tm value, or the point at which the fluorescence intensity increase / dt is the highest can be determined as the Tm value. In the case of using a probe that does not display a signal alone but shows a signal by hybrid formation, instead of the quenching probe, the amount of decrease in fluorescence intensity may be measured on the contrary.

From the Tm value, the genotype at the detection target site of the β2AR gene is determined. In Tm analysis, a hybrid (perfect match) that is completely complementary results in a higher Tm value indicating dissociation than a hybrid (mismatch) having a single base. Therefore, by determining the Tm values of the hybrids that are completely complementary to the probes and the Tm values of the hybrids having different monobases, the amplification products can be determined to match the probes or mismatches in advance. For example, assuming that the base of the detection target site in the detection target sequence is a variant (for example, guanine at the 4265th base in SEQ ID NO: 1), the probe complementary to the detection target sequence having the same (SEQ ID NO: 2). When y is cytosine), if the Tm value of the formed hybrid is about the same as the Tm value of the hybrid which is completely complementary, it can be judged that the polymorphism of the said amplification product is variant. If the Tm value of the formed hybrid is about the same as the Tm value of the other monobasic hybrid (lower than the Tm value of the completely complementary hybrid), the polymorphism of the amplification product is wild type (4265 in SEQ ID NO: 1). The first base is adenine). In addition, when only one Tm value is detected, it can be determined that it is heteroconjugation, when both homo-bonding property and both Tm values are detected. In this way, the polymorphism of the β2AR gene can be determined from the Tm value for the β2AR gene analysis probe.

According to the β2AR gene analysis method of the present invention, by using the probe of the present invention, as described above, the Tm value when the probe and the detection target sequence are a perfect match and the Tm value when the mismatch is sufficiently determined Indicates a possible difference. This difference is 4 degrees C or more, for example, Preferably it is 6 degrees C or more.

In addition, in the present invention, as described above, instead of the method of raising the temperature of the reaction solution containing the probe (heating the hybrid forming body) and measuring the signal fluctuation accompanying the temperature rise, for example, a hybrid The signal fluctuation at the time of formation may be measured.

Polymorphic Analysis Method

The polymorphism analysis method of this invention is a polymorphism analysis method which analyzes the polymorphism of the detection target site | part in the (beta) 2AR gene by analysis of melting temperature, It is characterized by including the (beta) 2AR gene analysis method of this invention. The polymorphism analysis method of the present invention can be carried out in the same manner as the β2AR gene analysis method of the present invention described above.

Next, the Example of this invention is described. However, the present invention is not limited by the following examples.

Example 1

Blood was collected from eight subjects using a heparin lithium blood collection tube (samples 1 to 8). 10 microliters of obtained blood and 90 microliters of distilled water were mixed, and 10 microliters of this liquid mixture and 90 microliters of distilled water were also mixed. 10 µL of these mixtures was added to 40 µL of the PCR reaction solution having the following composition, and PCR was performed using a thermal cycler. PCR conditions were 60 seconds at 95 degreeC, and 50 cycles were repeated for 1 second at 95 degreeC, and 10 second at 56 degreeC as 1 cycle, and 1 second at 95 degreeC, and 60 second at 40 degreeC. Subsequently, the PCR reaction solution was heated from 40 ° C. to 95 ° C. at a rate of temperature rise of 1 ° C./3 sec., And the change in fluorescence intensity over time was measured. The measurement wavelength was 450-480 nm (detection of fluorescent dye Pacific Blue), 515-555 nm (detection of fluorescent dye BODIPY FL), and 585-700 nm (detection of fluorescent dye TAMRA). In addition, the time required for 50 cycles of PCR was about 1 hour.

(Probe)

Probe for β2AR Gene

5'-cgcatggcttcCattgggtgcca- (Pacific Blue) -3 '(SEQ ID NO: 72)

Probe for β3AR gene 1

5'-cgtggccatcgccCggactc- (BODIPY FL) -3 '(SEQ ID NO: 83)

Probe 2 for β3AR Gene

5'-cgtggccatcgccCggactc-P-3 '(SEQ ID NO: 83)

UCP1 Gene Probe

5 '-(TAMRA) -cacagtttgatcGagtgcatttg-3' (SEQ ID NO: 95)

(Primer set)

β2AR gene F1 primer

5'-cccgggaacggcagcgcctt-3 '(SEQ ID NO: 9)

β2AR gene R1 primer mix

5'-cccacacctcgtccctttSctgcgt-3 '(SEQ ID NO: 18)

S is c or g, mixing both primers in a 1: 1 (molar ratio)

β3AR gene F2 primer

5'-ccaccgtgggaggcaacc-3 '(SEQ ID NO: 26)

β3AR gene R2 primer

5'-ctgcggccagcgaagtc-3 '(SEQ ID NO: 31)

UCP1 Gene F3 Primer

5'-ggaagacattttgtgcagcgatttctgattgacc-3 '(SEQ ID NO: 44)

UCP1 gene R3 primer mix

5'-cKtagcaaaggagtggcagcaagttctg-3 '(SEQ ID NO: 63)

K is g or t, mixing both primers in a 1: 1 (molar ratio)

The Tm value of the hybrid that matches the β2AR gene probe is 70.0 ° C, the Tm value of the hybrid that matches the mismatched probe is 62.0 ° C, the Tm value of the hybrid that matches the β3AR gene probe is 73.0 ° C, and the Tm value of the hybrid that misses is 66.0 ° C. The Tm value of the hybrid that matches the probe for the UCP1 gene is 65.0 ° C, and the Tm value of the hybrid that misses is 60.0 ° C.

The results of Samples 1 to 8 are shown in Figs. These figures are graphs of the Tm analysis showing the change in fluorescence intensity accompanying the temperature rise, and the derivative value on the vertical axis represents "-d fluorescence intensity increase amount / dt", and the horizontal axis represents temperature (hereinafter, the same). As shown in FIGS. 1-3, the polymorphism of the (beta) 2AR gene, (beta) 3AR gene, and UCP1 gene in each sample was determined from the peak of a signal. In order to support the results of these Examples, eight polymorphisms of the β2AR gene, the β3AR gene, and the UCP1 gene were confirmed by the RFLP method, and the same results as in Example were obtained. As described above, by using the primer set of the present invention, a β2AR gene, β3AR gene and UCP1 gene are simultaneously amplified in the same reaction solution using a whole blood sample that has not been pretreated, and the same reaction solution is used. Each polymorph of the gene could be interpreted.

Example 2

Blood was collected from three subjects using an EDTA blood collection tube (samples 1 to 3). 10 μL of the obtained blood was mixed with 70 μL of the following dilution A, and 10 μL of the mixed solution and 70 μL of the following dilution B were mixed. After 10 microliters of the obtained liquid mixture was heat-processed at 95 degreeC for 5 minutes, it was added to 46 microliters of PCR reaction liquids of the following composition, and PCR was performed using the thermal cycler. PCR conditions were 60 seconds at 95 degreeC, and 50 cycles were repeated for 1 second at 95 degreeC, and 15 seconds at 64 degreeC as 1 cycle, and 1 second at 95 degreeC, and 60 second at 40 degreeC. Subsequently, the PCR reaction solution was heated from 40 ° C. to 75 ° C. at a rate of temperature rise of 1 ° C./3 sec., And the change in fluorescence intensity over time was measured. The measurement wavelength was 450-480 nm (detection of fluorescent dye Pacific Blue), 515-555 nm (detection of fluorescent dye BODIPY FL), and 585-700 nm (detection of fluorescent dye TAMRA).

(Diluent A)

10 mM Tris-HCl (pH 8), 0.1 mM EDTA, 0.05% NaN ₃ , 0.3% SDS

(Dilution B)

10 mM Tris-HCl (pH 8), 0.1 mM EDTA, 0.05% NaN ₃

(Probe)

Probe for β2AR Gene

5 '-(Pacific Blue) -cccaatGgaagccatgc-P-3' (SEQ ID NO: 114)

Probe for β3AR gene 1

5 '-(BODIPY FL) -ctcggagtccGggc-P-3' (SEQ ID NO: 120)

Probe 2 for β3AR Gene

5 '-(BODIPY FL) -ctcggagtccAgg-P-3' (SEQ ID NO: 120)

UCP1 Gene Probe

5 '-(TAMRA) -cacagtttgatcGagtg-P-3' (SEQ ID NO: 139)

(Primer set)

β2AR gene F1 primer

5'-cgggaacggcagcgccttct-3 '(SEQ ID NO: 109)

β2AR gene R1 primer

5'-ccaccacccacacctcgtccct-3 '(SEQ ID NO: 134)

β3AR gene F2 primer

5'-gccaccgtgggaggcaacc-3 '(SEQ ID NO: 24)

β3AR gene R2 primer

5'-ggctgcggccagcgaagtc-3 '(SEQ ID NO: 28)

UCP1 Gene F3 Primer

5'-ggaagacattttgtgcagcgatttctgattgacc-3 '(SEQ ID NO: 43)

UCP1 gene R3 primer mix

5'-cKtagcaaaggagtggcagcaagttctg-3 '(SEQ ID NO: 63)

K is g or t, mixing both primers in a 1: 1 (molar ratio)

The Tm value of a hybrid that matches the β2AR gene probe is 58 ° C, the Tm value of the hybrid that misses a match is 51 ° C, the Tm value of the hybrid that matches the β3AR gene probe is 58 ° C, and the Tm value of the hybrid that misses is 51 ° C. The Tm value of the hybrid matching the probe for the UCP1 gene was 56 ° C, and the Tm value of the hybrid that missed was 49 ° C.

The results of Samples 1 to 3 are shown in FIG. 4. These figures are graphs of the Tm analysis showing the change in fluorescence intensity accompanying the temperature rise, and the derivative value on the vertical axis represents "-d fluorescence intensity increase amount / dt", and the horizontal axis represents temperature. As shown in Fig. 4, the polymorphisms of the β2AR gene, the β3AR gene, and the UCP1 gene in each sample were determined from the peaks of the signals. In order to support the results of these examples, the three subjects confirmed the polymorphisms of the β2AR gene, the β3AR gene, and the UCP1 gene by the RFLP method. As a result, the same results as in Example were obtained. As described above, by using the primer set of the present invention, a β2AR gene, β3AR gene and UCP1 gene are simultaneously amplified in the same reaction solution using a whole blood sample that has not been pretreated, and the same reaction solution is used. Each polymorph of the gene could be interpreted.

Example 3

Various probes for the β2AR gene were produced and analyzed for Tm. First, the probe shown in following Table 9 and the oligonucleotide (complementary strand DNA) shown in following Table 10 complementary to each said probe were synthesize | combined. In the probe sequence of Table 9 below, the base of the capital letter is the base corresponding to 4265th in SEQ ID NO: 1, and these probes are antisense complementary to the 4265th (G, A) or the same in the sense chain. It was designed as a probe for detecting bases (C, T) in the chain. The oligonucleotide shown in following Table 10 is a partial sequence of the sense chain or sense chain of (beta) 2AR gene, and is a sequence containing the 4265th base of sequence number 1 or the base complementary to it. And using these probes and complementary strand DNA, the reaction liquid was prepared so that it might become the composition of Table 11 below. When the detection target chain of the probe was a sense strand, the reaction solution to which the respective additions were made using 1A (4265A) and 1B (4265G) as complementary strand DNA. In addition, when the detection target chain of a probe was an antisense strand, the reaction liquid which added each was prepared using 2A (4265A) and 2B (4265G) as complementary strand DNA. These reaction solutions were heated using a thermal cycler (brand name Smart Cycler, manufactured by Cepheid) at a rate of temperature rise of 1 ° C./3 sec., And the reaction solution was heated from 45 ° C. to 95 ° C. over time, and the fluorescence intensity over time. The change of was measured. The measurement wavelength was 565-605 nm (detection of TAMRA, ch3).

The result using the probe of Example 3-1 is shown in FIG. 5, and the result using the probe of the comparative example 3-9 and the comparative example 3-11 is shown in FIG. These figures are graphs (fusion curves) of the Tm analysis showing the change in fluorescence intensity accompanying a temperature rise, and the derivative value on the vertical axis represents "-d fluorescence intensity increase amount / dt", and the horizontal axis represents temperature (hereinafter, the same). ). In Fig. 5, a melting curve (thin line) of a system to which the complementary DNA (1A) is added to the reaction solution and a melting curve (thick line) of the system to which the complementary DNA (1B) is added to the reaction solution are shown. 6 shows a melting curve (a thin line) of a system to which the complementary DNA (2A) is added to the reaction solution and a melting curve (a thick line) to the system to which the complementary DNA (2B) is added. Illustrated.

As shown in FIG. 6, when the probe of Comparative Example 3-9 was used, a signal peak of 4265G was not detected. In the case of using the probe of Comparative Example 3-11, the Tm value (68 ° C) of the peak representing the perfect match between the probe and the complementary DNA (2B) and the mismatch between the probe and the complementary DNA (2A) were determined. The difference of Tm value (65 degreeC) of the peak shown was small (3 degreeC). For this reason, from the melting curve using the probes of these comparative examples, it was difficult to judge which of the perfect match and the mismatch was shown with excellent reliability. Moreover, also about the other comparative example, the melting curve similar to the comparative example 3-9 or the comparative example 3-11 in FIG. 6 was shown. In contrast, in the case of using the probe of Example 3-1, as shown in FIG. 5, a signal peak indicating a perfect match between the probe and the complementary DNA (1B), and the probe and the complementary DNA (1A) Both of the signal peaks indicating a mismatch could be fully confirmed. The difference between the Tm value (75 ° C) of the peak representing the perfect match and the Tm value (68 ° C) of the peak representing the mismatch was about 7 ° C, which was a sufficient difference to discriminate both. For this reason, according to the probe of an Example, it is possible to fully judge which of a perfect match and a miss match is shown.

Example 4

The human purified genome which shows the homozygous whose 4265th in SEQ ID NO: 4265 is 4265A / 4265A, and the human purified genome which shows the homozygote of 4265G / 4265G were prepared. 1 µL of each of these purified genomes was added to 24 µL of a PCR reaction solution having the following composition, respectively, and PCR was performed using a thermal cycler (trade name Smart Cycler, manufactured by Cepheid). The PCR conditions were repeated 60 cycles at 95 ° C for 1 second at 62 ° C and 30 seconds at 62 ° C for 50 cycles. Subsequently, the reaction solution was heated from 45 ° C to 95 ° C with a rate of temperature rise of 1 ° C / sec, and the change in fluorescence intensity over time was measured. The measurement wavelength performed Tm analysis. The detection wavelength was set to 505-537 nm (detection of fluorescent dye FAM, 1ch).

(Probe)

Probe for β2AR Gene Analysis

5'-cgcatggcttcCattgggtgcca- (BODIPY FL) -3 '(SEQ ID NO: 72)

(Primer set)

Forward primer for β2AR gene

5'-gggaacggcagcgccttcttgct-3 '(SEQ ID NO: 165)

Reverse primer for β2AR gene

5'-tggtgaccgtctgcagacgctcgaac-3 '(SEQ ID NO: 166)

The region amplified by PCR using the primer set is a DNA chain of about 200 bases including the 4265th sequence in SEQ ID NO: 1. From the genome representing 4265G / 4265G, the DNA chain with the 4265th G (antisense chain C) was amplified, and from the genome representing 4265A / 4265A, the DNA with the 4265th A (antisense T) was amplified.

The Tm value of the hybrid that matches the β2AR gene analysis probe shown in SEQ ID NO: 72 is 69 ° C, and the Tm value of the hybrid that misses is 62 ° C.

These results are shown in FIG. Fig. 7 is a graph (melting curve) of the Tm analysis showing the change in fluorescence intensity with increasing temperature, the derivative value on the vertical axis represents "-d fluorescence intensity increase amount / dt", and the horizontal axis indicating temperature (hereinafter, the same) . In Fig. 7, the melting curve (thin line) of the system which added the human purified genome which shows 4265A / 4265A homozygotes, and the melting curve of the system which added the human purified genome which shows 4265G / 4265G homoconjugate (thick line) Together).

As shown in FIG. 7, in the coexistence of the probe of Example 4, DNA in the β2AR gene of the human purified genome was amplified and Tm analysis was performed, and as a result, it was possible to distinguish and detect the 4265th base. Specifically, Tm value (69 degreeC) of the peak which shows the perfect match between the said probe and the amplification derivative from 4265G / 4265G homoconjugate, and the peak which shows the mismatch between the said probe and the amplification derivative of 4265A / 4265A homoconjugate. The difference in Tm value (62 占 폚) was about 7 占 폚, which was sufficient to discriminate both. For this reason, according to the probe of the Example, it is possible to fully analyze the 4265th polymorphism in (beta) 2AR gene.

As mentioned above, according to the primer set of this invention, the area | region containing the site | part which generate | occur | produces a specific polymorphism in the obesity gene ((beta) 2AR gene, (beta) 3AR gene, or UCP1 gene) can be specifically amplified with high efficiency. For this reason, unlike the conventional method mentioned above, time and cost can be reduced. In addition, since the region including the polymorphism detection site is specifically amplified in this manner, for example, by using a probe complementary to the detection target sequence including the detection site, Tm analysis is performed as it is using the reaction solution. By doing this, it is possible to type the polymorphism. In addition, since amplification and typing can be performed with one reaction solution, the operation can be automated. In addition, if the primer set of the present invention is used, for example, even if a sample containing a contaminant (for example, whole blood, oral mucosa, etc.) can be omitted, the amplification reaction can be performed more quickly and simply. In addition, when the primer set of the present invention is used, the amplification reaction can be performed at an amplification efficiency superior to that of the prior art, so that the amplification reaction can also be shortened. Therefore, according to the primer set of the present invention, the reagent containing the same, and the method for producing an amplification product using the same, it can be said that the polymorphism of the obesity gene can be interpreted quickly and simply, which is very effective in the medical field.

                         SEQUENCE LISTING <110> ARKRAY, Inc. <120> Primer set and agent for amplification obese gene and use <130> TF07041-01 <150> JP2006-322961 <151> 2006-11-30 <150> JP2006-322960 <151> 2006-11-30 <150> JP2007-232614 <151> 2007-09-07 <160> 167 <170> PatentIn version 3.1 <210> 1 <211> 9968 <212> DNA <213> Homo sapiens <400> 1 tgcaaaagac atgaagtcat ccttttttat ggctgcacag tattccatgg tgtatatgtg 60 ccacattttc tttatcaagt ctattcttga tgggcatttg ggttggttcc aagtctttgc 120 tattgtgaac agtgctgcaa taaacataca tgtccatgtg tctttatagt agaatgattt 180 ataatcctta gggtatatac caggtaatgg gattgctggg tcaaatggta tttcttgttc 240 tagatccttg agggattgcc acactgtctt ccacaatgtt tgaactaatt tacattccta 300 ccaacagtgt aaaagtgttc ctatttctcc acatcctttc cagcatctgt tgtttcctga 360 cttcctgttt tttttttgag acggagtctc actgtgtcac cccggatgga gtgcagtggc 420 acaatctcgg ctcactgcaa cctccacctc ccaggttcaa gcgattctcc tgtttcagcc 480 cccagagtag gtgagactac aggcatgcca caacttctgg ctaatttttg tatttttatt 540 agagacgaag tttcaccatg ttggtcaggc tgctctcgaa gtcctgacct caggtgatcc 600 acccacctca gcctcccaaa gtgctgggat tacaggcatg agccactgcg cccggtctgt 660 ttcctgatgt gttaatgatc gccattgtat ctggtgtgag atggtatttc attgtggttt 720 tgatttgcat ttcactaata accagtgctg atgatctttt cttcatatgt ttgttggctg 780 cattaatgtc ttcttttgag aagtgtctgt tcatatcctt tgcccacttt ttcatggggc 840 tgtttgtttt ttcttgtaaa tttgtttaag ttctttgtag attctggata ttagcccttt 900 gtcagatgga tagattgcaa aatttttctc ccattctgta ggttgcctgt tcaccctgat 960 gatagtttct tttgctgtgc agaaactctt tagtttaatt agatcccatt tgtcaatttt 1020 ggcttttgtt gccattgctt ttgatgtttt agtcatgaag tctttgccca tgcctatgtc 1080 ctggatgtta ttgcctagat tttcttctag ggtttttatg gttttaggtc ttacgtttaa 1140 gtctttaatt catcttgagt taattcttgt ataaggtgta aggaaggggt ccagtttcag 1200 ttttctgcat atggctagcc agtccttctt gatttagtat ttgtgggttt taaaaaagga 1260 gtttcccaaa atattcagtt aaacttttaa gtgacttacg tgtatatcta aatacatgat 1320 cagttaatat ttgtcttaaa ggggttttct ttgttctttt cttattatag gaaggttaaa 1380 caatatgctt atttatgcca tagcttcaca aacaggaagg aggttttaaa tggtttagtt 1440 ccacaatttg agtagatgca tatttaaaga aacgttgttg cataataaat actgcctctt 1500 cctaaaatgc atcatgccac agccaatttt ggaaaacaca aatatgaggt gagtgtattt 1560 tgaaaactat gtgaatataa tagatcctta attcatattt gtggatttta tgggaaatac 1620 ttgttttcta aggcatctgt cttgcaaaaa gtcagtttct gctatgaagg atgttaaagg 1680 ggatatgtag gttaaattct gtttctgagc tttgcttcca gagtaaacac ccaacttact 1740 tttgccctaa agtattttat tgttctagta gagaagacta acaacatatt ctaaaccact 1800 aagtaattta tgtaaacttc gcttacaaac tatacttgtg tgacacttat atgagcaaaa 1860 gcattttcat atttcttact atatcattca attcttgctt accccaatgg aagtgacttt 1920 atgccccttt agagacaatg gaaatcaggt acttcgtgat ttctcttaaa aaaaaaaaaa 1980 atgaactaga aagctccaag tttggtgaat ctggaacctg ggtattccag ttccagttgt 2040 agcccttcct ccctatccat cactcctgtc tgcatgtaat tatgcaatac attgaaaaga 2100 ttaaaagatg ggtcttggac tcaggcagac ctgggtcaaa tccagattct ggcactgccc 2160 agccattgcc cctgggcaag ccattttcct ctttgaacct catttgtgaa ttaagctaaa 2220 aatagtcccc acccccatgg gactgtggga aggattaaat agaataatgc atgaaaagca 2280 aatagcagaa tggtccataa atgttaacca ttgttatgtt attatgtaat ctacaaagta 2340 cgtttagtta cacttcatga aatactttca gtttttcaaa gacaccacta atacatggga 2400 aatcaaaccc tgaaaattaa tttcacttta gcagtaaagt cacatgccag atggaaagga 2460 tagtatttca tgaacaaaga tcttactttt gagatttggt cttacttttt tctttttctt 2520 aagggagaat tatcttgtgt tttttgtttt gttttgtttt gagatggagt cttgttctgt 2580 cacccaggct ggagcgcagt gacgtgatct cggctcactg caaccttcac ctcccgggtt 2640 caagagattc tcctgtctca gcctcccgag tagctgggac tacaggtacg tgccaccaca 2700 cctggctaat ttttgtattt ttagtagaga caagagttac accatattgg ccaggatctt 2760 ttgctttcta tagcttcaaa atgttcttaa tgttaagaca ttcttaatac tctgaaccat 2820 atgaatttgc cattttggta agtcacagaga gccagatggt ggcaatttca catggcgcaa 2880 cccgaaagat taacaaacta tccagcagat gaaaggattt tttttagttt cattgggttt 2940 actgaagaaa ttgtttgaat tctcattgca tctccagttc aacagataat gagtgagtga 3000 tgccacactc tcaagagtta aaaacaaaac aacaaaaaaa ttaaaacaaa agcacacaac 3060 tttctctctc tgtcccaaaa tacatacttg catacccccg ctccagataa aatccaaagg 3120 gtaaaactgt cttcatgcct gcaaattcct aaggagggca cctaaagtac ttgacagcga 3180 gtgtgctgag gaaatcggca gctgttgaag tcacctcctg tgctcttgcc aaatgtttga 3240 aagggaatac actgggttac cgggtgtatg ttgggagggg agcattatca gtgctcgggt 3300 gaggcaagtt cggagtaccc agatggagac atccgtgtct gtgtcgctct ggatgcctcc 3360 aagccagcgt gtgtttactt tctgtgtgtg tcaccatgtc tttgtgcttc tgggtgcttc 3420 tgtgtttgtt tctggccgcg tttctgtgtt ggacaggggt gactttgtgc cggatggctt 3480 ctgtgtgaga gcgcgcgcga gtgtgcatgt cggtgagctg ggagggtgtg tctcagtgtc 3540 tatggctgtg gttcggtata agtctgagca tgtctgccag ggtgtatttg tgcctgtatg 3600 tgcgtgcctc ggtgggcact ctcgtttcct tccgaatgtg gggcagtgcc ggtgtgctgc 3660 cctctgcctt gagacctcaa gccgcgcagg cgcccagggc aggcaggtag cggccacaga 3720 agagccaaaa gctcccgggt tggctggtaa gcacaccacc tccagcttta gccctctggg 3780 gccagccagg gtagccggga agcagtggtg gcccgccctc cagggagcag ttgggccccg 3840 cccgggccag cctcaggaga aggagggcga ggggagggga gggaaagggg aggagtgcct 3900 cgccccttcg cggctgccgg cgtgccattg gccgaaagtt cccgtacgtc acggcgaggg 3960 cagttcccct aaagtcctgt gcacataacg ggcagaacgc actgcgaagc ggcttcttca 4020 gagcacgggc tggaactggc aggcaccgcg agcccctagc acccgacaag ctgagtgtgc 4080 aggacgagtc cccaccacac ccacaccaca gccgctgaat gaggcttcca ggcgtccgct 4140 cgcggcccgc agagccccgc cgtgggtccg cctgctgagg cgcccccagc cagtgcgctt 4200 acctgccaga ctgcgcgcca tggggcaacc cgggaacggc agcgccttct tgctggcacc 4260 caatggaagc catgcgccgg accacgacgt cacgcagcaa agggacgagg tgtgggtggt 4320 gggcatgggc atcgtcatgt ctctcatcgt cctggccatc gtgtttggca atgtgctggt 4380 catcacagcc attgccaagt tcgagcgtct gcagacggtc accaactact tcatcacttc 4440 actggcctgt gctgatctgg tcatgggcct ggcagtggtg ccctttgggg ccgcccatat 4500 tcttatgaaa atgtggactt ttggcaactt ctggtgcgag ttttggactt ccattgatgt 4560 gctgtgcgtc acggccagca ttgagaccct gtgcgtgatc gcagtggatc gctactttgc 4620 cattacttca cctttcaagt accagagcct gctgaccaag aataaggccc gggtgatcat 4680 tctgatggtg tggattgtgt caggccttac ctccttcttg cccattcaga tgcactggta 4740 ccgggccacc caccaggaag ccatcaactg ctatgccaat gagacctgct gtgacttctt 4800 cacgaaccaa gcctatgcca ttgcctcttc catcgtgtcc ttctacgttc ccctggtgat 4860 catggtcttc gtctactcca gggtctttca ggaggccaaa aggcagctcc agaagattga 4920 caaatctgag ggccgcttcc atgtccagaa ccttagccag gtggagcagg atgggcggac 4980 ggggcatgga ctccgcagat cttccaagtt ctgcttgaag gagcacaaag ccctcaagac 5040 gttaggcatc atcatgggca ctttcaccct ctgctggctg cccttcttca tcgttaacat 5100 tgtgcatgtg atccaggata acctcatccg taaggaagtt tacatcctcc taaattggat 5160 aggctatgtc aattctggtt tcaatcccct tatctactgc cggagcccag atttcaggat 5220 tgccttccag gagcttctgt gcctgcgcag gtcttctttg aaggcctatg ggaatggcta 5280 ctccagcaac ggcaacacag gggagcagag tggatatcac gtggaacagg agaaagaaaa 5340 taaactgctg tgtgaagacc tcccaggcac ggaagacttt gtgggccatc aaggtactgt 5400 gcctagcgat aacattgatt cacaagggag gaattgtagt acaaatgact cactgctgta 5460 aagcagtttt tctactttta aagacccccc cccgcccaac agaacactaa acagactatt 5520 taacttgagg gtaataaact tagaataaaa ttgtaaaatt gtatagagat atgcagaagg 5580 aagggcatcc ttctgccttt tttatttttt taagctgtaa aaagagagaa aacttatttg 5640 agtgattatt tgttatttgt acagttcagt tcctctttgc atggaatttg taagtttatg 5700 tctaaagagc tttagtccta gaggacctga gtctgctata ttttcatgac ttttccatgt 5760 atctacctca ctattcaagt attaggggta atatattgct gctggtaatt tgtatctgaa 5820 ggagattttc cttcctacac ccttggactt gaggattttg agtatctcgg acctttcagc 5880 tgtgaacatg gactcttccc ccactcctct tatttgctca cacggggtat tttaggcagg 5940 gatttgagga gcagcttcag ttgttttccc gagcaaagtc taaagtttac agtaaataaa 6000 ttgtttgacc atgccttcat tgcacctgtt tctccaaaac cccttgactg gagtgctgtt 6060 gcctccccca ctggaaaccg caggtaacta cttgtaatta ctgcccatga cttaatgtag 6120 aatgatacaa gaatgacatg cacagattgc ttaacccttt catttgcctt tgagtctgct 6180 gctgcaaagc tgcatctctc ctgacacttg tgccccaaat cagttctgcc tgctcttagt 6240 atagctcaac tctccctatg gttattgttc tgtgttgtta cctcagaaac actgactcac 6300 agaagcggag ttaaggggat atgttttttt ctctccacgt gcacccacca cccaccttcc 6360 agttctactt gtttcaaaac tgtttatatt tctgtcttgg ccatgtgtta cagtggagct 6420 ctttgtactg catcagggct tggcatttta gggataagga agatgttctt atgaggaagc 6480 tactcagaca tggccccgta attctgaggg aaaattcaaa aggcattggt catggggaga 6540 aaagctggag aacacataac tgatggatac ctcatgaact agaaacagaa ttttaacccc 6600 ttttccttct ttcctttggt ccctgttttc ttctcccact gactctcctc gattcagtgt 6660 aaaccaaggt tctgagtctt agcactgtta gcattttgga ccagataact ctttgttatg 6720 ggggctgcat cattgtatgt gtagcacctc tggcctctgt tcattagatg ccaatagcac 6780 cccctgctta taacaagcaa aaatgtttcc agacattgca aaagagcccc tgaggtgcga 6840 attagcccct ggttgagagt cactggtaga aactgtaaaa atctcagcag atacatacat 6900 tctttctaat gcaagcgctt gattgtgcag agccttagag agggattttc acagttcacc 6960 taggcagtaa cagaccctca ccagcactct ttccattcca tcatgctgcc ttctaaactt 7020 gttttctagc tgcccaaata gtgatcatga aatgttaaga aggctttaag tctgtacatg 7080 aattgtttga gagggtttat caatggaggt gaggcctgtg ggccatgact cctgtttgtg 7140 aagagattat aatactgtca agaggcacgt taggggaaat cacaaaagta aacacatttc 7200 ttctcccagc ccctttctat ttttgcctgt gtgtctgagc cagagcttgg cccaggtttg 7260 atgaagtgga tcgtcctcct tggcaacgcc aggctagagc agatcagcct gcagggttca 7320 ttgccattcc actggctcat gaagctgact ccactcccct cttcctttct gttgcagcca 7380 aggtccccaa ccagaaaagc attggccttc tctgcttcct gtcaactcaa tgatgggatg 7440 tttgggtgag caccgagcta tcaggagaag gttaggcgcc tgtgattttg gaacatgcca 7500 tggcaaattg gagaatgtgt gtcattcagt gcttttactt ttttccaagg gttttcatac 7560 ctattgaaaa cccttattac acattatcac cttctctttc tactgctatt atcacattct 7620 ctttctactg ctctggtctc cacactcaga gatttgggca gcttctttgg ctaatattca 7680 tgctccctgt agcctcataa gatctcagac atggaagagc ccatagaaag tatttaacat 7740 ctgatgagac tgaaacgctg tgaggtgaag ggcttgccca aggtaagcag ccaaggatgt 7800 cagagtggga ctcaagccag ggaacccaac tgctattcca ggaactgctg cattctctcc 7860 accacattag cattgcgttc tttcctcacc ctcaactggg gctataacat aacacattca 7920 tttcagccaa tatatttttc ttttgtcctt aacacaaaat ttagagcata aacaaataat 7980 ctgcaataga gacaaaagaa ataattgttc atttaactca acaagcatcc actaggatat 8040 cttctgcatt tagggaagct gggagattcc ttagctcttt ataaccagat gtcggtgttg 8100 tatatatact cttttgccta aaggaatgtc taatgtaatt tctgttaaaa ttcaggtatt 8160 aatgttaatt cagattccca gctaaaagga gagtttaacc atattcacgt tcctttagga 8220 atactgtaga cacaagaacc ttgattagtt ttaagggtcc tgataagcaa gagcattcta 8280 ggcatatctt aatcctttgc tttctacctc tttggtgtgt tgctttgttt cttttgaggg 8340 gttggctttt gtcagtgtcc cttcgtctct ctgtttgctg acatgctggc cacctaaggt 8400 ttgtgttgta ttcctcatcg tgagtttttt ttttgcctgg acagcaagtt ctcagagtct 8460 gtcaaataag aagaactttt ttctaagatg caagctgaga ggtgtgaaca gtggcaggac 8520 agggtgagcc tccccactgc aataattaat gggataagga atctggagaa aggggagctt 8580 gagaatagga actgtcttta catgattctt agaatgtttc ttatggtgaa cctattgcca 8640 aatggagcct aaaccagaat cagtcagaaa gtatttatgg agcacctact gtatgcagca 8700 tgagaaaagt ataggtttcc atttctgcct tttggatcat gatattaagg atactaagca 8760 gatacctttt atggaatctt actaaaagtt agtgactatg ttagacacct gttaggcgtt 8820 atgtcctcac aactgtatga tgtaggtagc ataattgtcc tcatctaaca aatgagggag 8880 ctgaggctca gaaagcttca gtaactttcc aaagccatac aaccaactag ttgcagagtc 8940 aggacgagaa cccaggtctc tgtgattcca ggatccatgg agcctagcac ccaggcaaac 9000 caggaagcag cacgaggtag cttagaatct gtgccagaac aagtgtaaga actggaaatg 9060 caatactttg ttgagagaag acggagactg ctgtgggttg aggcagggag gaatgttccc 9120 ttccaagcat gtgactgcag ggtttctggg gactgacaca aaaaaccaat cactgaagtg 9180 cctagtttgc ttctccaagt ctttgttttg tcttctcatc agagaatcag accaaaatgg 9240 aaagaggata tgaaactcta aaacgaacac agcagagtaa agcagaaaca caggctgccc 9300 tacgtcctgg cactctttct cagctccaaa gttgggaagg cctcctaaat tgagtgggta 9360 gccagacctt atggaaattt gataggcctg ggtagccata taggtatgtt ttccatcttg 9420 agctagcatt ctagatcaaa ggagatgatt ttctgacagc agagagcaag gaagcttaat 9480 atgctttgaa tgatactttc cattgacagt ataacctcta ctaaattcag tctgtgctaa 9540 tccatacttc actgacagtg caaatataat ttgaaggagg gatttttcta aatgcgtaag 9600 agaacaaact tcctaagcac tttcaagaac ttgagaaatt ctggtgtttt gtgaataata 9660 ggaggcaggg tgaggtgaaa agagcccccc tccaacctcc cacccaccac cccactctct 9720 ctctctctga agttgggtga ggcggttttc ttcagccgac ttccgtttac tcatctgtaa 9780 tttgaggcca gtacaggtgg tatctaagct ctgctcccct ctgaatttat acagttgtta 9840 atttacaggc tagagataat catttatatt tctaattaga gttgatgaca tttgccatat 9900 aaacaatggt tgaatattct gaaggttgtc agcctgcaga agagaatgct gtgaagtact 9960 atttggtg 9968 <210> 2 <211> 11265 <212> DNA <213> Homo sapiens <400> 2 acttttgtat atctatctgg aaggatcaca gatacatagt caaattaaaa taagcaaggt 60 gcagaacagt gggtatatat gactgctatt tgtatagaaa agaaaggata aaagaatata 120 tatatttgta ttgacttgta tctatataaa atatgagccg agggataaac cagagactaa 180 tggtagttac ctgctaggga gatgaaaact gggatgaagg caagtctcta ttatatgcat 240 ttgttatatt ttgatggttg aataatgtgc acgtataatc tattcaaaat attaagttat 300 aaaacacaaa atcacaaaag agacccaata gtctatagac taaggtggca aaagagggag 360 cccattcccc ctgccatctg cttgggcggg ggaaaaccag actgaggaat gagagcaggg 420 atgagttggc agaggaggaa gaagccaagt gtctcaacat tgatggattt gctcataaaa 480 ccatccctaa ccggagtcag aaatttctct tcttacagtg tcgtttggtt attggcctaa 540 tcattcccca aactcaggca cactcaggtg gtttgagaaa tcctgtcttt tttgaaaatg 600 ttaaacattt ttacacactt cttccaaatt acaaggtcac ccctaaggca ttgacatcat 660 tgaggttgct tcactttccg gcaccccttt cccttccccc tccaatgctg atgtcctgga 720 aaaaaattgc tgaatggcat gcattgattt tggagattac tttttgggaa tcttatgata 780 tccctttgag ataactggca acaacactct ggggtgctgc aaaaccctac ttgggagtga 840 ctgccttgct ggtcaacagg aagccacagc caagactctg tgcccagtgg ggcctgcacc 900 actctgtcat gacttgagtg ggggcagtaa gaggtggaag gggccagagg ctgcctcttc 960 actttcaccc ccatggattt ccatggatcc aaaggcctgc ttggggttct tcatgggcat 1020 agaagtcact tgcttgggag cccaagacaa accctgctca tgggtgagca gctgggtccc 1080 tgggagacga ggctggttct ttttccttgg ggataatttt aggttctgaa ttcccctgca 1140 accttcctaa ctcccagccc ccagctcagc ctcccagagg gattgttcat accagtgact 1200 ataaagtgtt ctctcgaaat atagagctgt tcagagacct gccagggcat agccctggag 1260 gcattttcca tgataatttt tccccctttg tcccattgtt tcagaggagg aagcatgata 1320 gctcaagaat caggtcctga tagtccctga taaggtgtca catcttcctg cctctccact 1380 tgaaccctgg ggctttattt tccagcgccc ccctccccgt acgcaatctg agaggaactg 1440 gtttagagat tgagagagca ggggctggaa aaaggaagcc ttgcaagtct tgagtgaggg 1500 cagggagaag ggaagaggaa gagggaagcc tctggtggaa ccgcctaaga gacttcagag 1560 gctttgaaac accaggcctg ccaccacaca cgctcaaaaa tgaacgcatg aattcctcaa 1620 ctcaacgcct tctagttttt aaaagttaat gaatgaaatc cttaaggaag tccggacacg 1680 gtgactcaca cctgtaatcc cagcattctg ggaggccgag gcgggtggat catctgaggt 1740 caggagtttg agaccagcct ggccaacatg gtgaaacccc gtctctacta aaaatacaaa 1800 aatttgctgg gcttggtggc acatgctcat cccagctact tgggtggctg ggaggcttgg 1860 gaggctgagg cagaagaatt gcttgaacct gggaagtgga ggttgcagtg agcagagatt 1920 gtgccactgc actccagcct gggcaacaag agcaaaattt catctcaaaa aacaaataaa 1980 caaacaaaaa acctaaagga agtggagcga acacttacca ttggggagat ggcaatggaa 2040 gagttgtgta tagtttgggg ggatggtgga cctgcctcat acccagtcac ccatttaccc 2100 attttttttt ttaaaatagg gtcttgcttc ttgccgtctt ccaagctgga gtgcagtagt 2160 agcatgatca cagatcactg taaccttgaa ctcctgggct caagcgatcc tctcctctca 2220 gcctcttgat tcatcctttc tttttttttt tttttttttt ttttctgata tggagtttcg 2280 ctcttgttgc caaggctgga gtgccatggt gtgatctcgg ctcactgcaa cctctgcctc 2340 ccaggttcaa gagattctcc tgcctcagcc tcttgagtag ctgggattac aggcatgcgc 2400 caccacacct ggctaatttt tttttttttt ttttgtattt ttagtagaga cagggtttct 2460 ccttgttgtt caggctggtc ttgaactcct gacctcaggt gatctgccca cctcgacctc 2520 ccaaagtgct gggattacag gcgtgagcca ccgcgcccac ctgttgattc atccattttt 2580 aatatgtctt tatagcagaa agattttaag agaaggggct atctagaaaa tctctaggtg 2640 gaaaggtgca tggaaggggc agtgcagagt ggggaagatg gagaaattga aattcttcag 2700 catcccagac tacatagtgc caaatccaaa tgcaaatcca gagctagcaa tgaaaaagac 2760 tcctggccgg gcgcgatggc tcacgcctgt aatcccagca ctttgggagg ccgaggcggg 2820 cggatcacga ggtcaggaga tcgagaccat cctggccaac atggtgaaac cccgtctcta 2880 ctaaaaatat aaaaattagt tgggcatgct ggcgggtgcc tgcagtccca gctacttggg 2940 aggctgaggc aggagaagcg cttgaacccg ggaggtggag attgcagtga gcagacatca 3000 catcattgca ctccagcctc cagcctgggt gacagagcga gacgccgtct caaaaaaaaa 3060 aaaaaaaaga aaagaaaaag aaaaagactc ctgagcctcc tagtggttca cgcctgcaat 3120 tccaactact caggaggctg ggacaggagg gtggcttgag cccgggagtt caaagccagc 3180 ctgggaaaaa tagagagaga gagagagact tgaaatatgg taaatttcct cagtacctga 3240 aaatattaaa aaaaacaaaa agctagaagg aaagagaagg taactgaaac agaagggaaa 3300 ggggacagat ctcaccaagc tgaggtcttg ggagaggaga tactggctga gccctattac 3360 ttaatttaaa ataccttagg ggaggccacc caagtggatg cggggctcct gtgaatcctt 3420 tgcttgactc cagcgggtta cctttgcctc tgatacataa agggtgggga tgggagcgct 3480 ctcctctctc cttcccctgc cttgctgtgg gaacttctgg gaaaggaggt gcagggctcc 3540 aggaagccag tgcccaggga gtgctatgct gagtccagga gcctggccac ggcaggggtg 3600 gacagatggt ggcagaggaa ccacggtgtc ccttcctcca gatttaagct aaaggaaacg 3660 tggagcatcc cattggccat cctccccact ctccaattcg gctccagagg cccctccaga 3720 ctataggcag ctgccccttt aagcgtcgct actcctcccc caagagcggt ggcaccgagg 3780 gagttggggt ggggggaggc tgagcgctct ggctgggaca gctagagaag atggcccagg 3840 ctggggaagt cgctctcatg ccttgctgtc ccctcccctg agccaggtga tttgggagac 3900 cccctccttc cttctttccc taccgcccca cgcgcgaccc ggggatggct ccgtggcctc 3960 acgagaacag ctctcttgcc ccatggccgg acctccccac cctggcgccc aataccgcca 4020 acaccagtgg gctgccaggg gttccgtggg aggcggccct agccggggcc ctgctggcgc 4080 tggcggtgct ggccaccgtg ggaggcaacc tgctggtcat cgtggccatc gcctggactc 4140 cgagactcca gaccatgacc aacgtgttcg tgacttcgct ggccgcagcc gacctggtga 4200 tgggactcct ggtggtgccg ccggcggcca ccttggcgct gactggccac tggccgttgg 4260 gcgccactgg ctgcgagctg tggacctcgg tggacgtgct gtgtgtgacc gccagcatcg 4320 aaaccctgtg cgccctggcc gtggaccgct acctggctgt gaccaacccg ctgcgttacg 4380 gcgcactggt caccaagcgc tgcgcccgga cagctgtggt cctggtgtgg gtcgtgtcgg 4440 ccgcggtgtc gtttgcgccc atcatgagcc agtggtggcg cgtaggggcc gacgccgagg 4500 cgcagcgctg ccactccaac ccgcgctgct gtgccttcgc ctccaacatg ccctacgtgc 4560 tgctgtcctc ctccgtctcc ttctaccttc ctcttctcgt gatgctcttc gtctacgcgc 4620 gggttttcgt ggtggctacg cgccagctgc gcttgctgcg cggggagctg ggccgctttc 4680 cgcccgagga gtctccgccg gcgccgtcgc gctctctggc cccggccccg gtggggacgt 4740 gcgctccgcc cgaaggggtg cccgcctgcg gccggcggcc cgcgcgcctc ctgcctctcc 4800 gggaacaccg ggccctgtgc accttgggtc tcatcatggg caccttcact ctctgctggt 4860 tgcccttctt tctggccaac gtgctgcgcg ccctgggggg cccctctcta gtcccgggcc 4920 cggctttcct tgccctgaac tggctaggtt atgccaattc tgccttcaac ccgctcatct 4980 actgccgcag cccggacttt cgcagcgcct tccgccgtct tctgtgccgc tgcggccgtc 5040 gcctgcctcc ggagccctgc gccgccgccc gcccggccct cttcccctcg ggcgttcctg 5100 cggcccggag cagcccagcg cagcccaggc tttgccaacg gctcgacggg taggtaaccg 5160 gggcagaggg accggcggct cagggtcggg aagcatgcga tgtgtccgtg ggtcaacttt 5220 ttgagtgtgg agtttattaa gagaaggtgg gatggctttg cttggagaga aaagggaacg 5280 aggagtagcg aaccaaaatg ggacccaggg tccttttctt tccggatcca gtcactaggg 5340 tagaagcaaa ggagggcgag cgggccgtcg ttcctcaccc aaggacccaa ggtgcgccac 5400 cggaaagcgc tgcggtgtcc cgaggactct cgcctcgcct ggtcggcttt agggattttt 5460 ttttttttta aatagagaca gggtttcgtc tctgtcgccc acgcgggaat gcagtggcgc 5520 gatctcagct cactgcagtc ttgaactcct ggctcctggg ctcaagcgat cctcccacct 5580 cagcctcctg agtatctggg actacaggcg agccccacca atcccagcta tttttaaaat 5640 ttcttgtaga gatggggtct tgctatgttg cccaggcttg tcttgaactt ctggcctcaa 5700 gtgatccttc tgcctcagcc ttccaaagca ttaggattac aggccggagc cagggcgccg 5760 ggtcggctct agttttggtt ttccagctca gttctttgcc cccctccccc gatttcttgc 5820 catcactaga cctggctcgg acttgaaggc agggctagtg cccccccacc cgccccccaa 5880 gccctcggcc tcagttctgg gttttctcaa aggtttgaca gctgtggagg tgagaatcca 5940 cttccggtat gaagtacagt tgtgagtgag gagcctgtga gtgcagatgt gtgccctccc 6000 gctccctggg ctgggttgga gtagggatgg ggtggggcgt gtgtggctgg gtggtgccct 6060 ggcgtttttg tgtaactaaa tatgcgttcc agggtctctg atctctgtca ttcccctcag 6120 tgcacctgtt gctcctttca ccccagggtc tattatctcc actttttttc ccagggcttc 6180 ttggggagtt tcttaggcct gaaggacaag aagcaacaac tctgttgatc agaacctgtg 6240 gaaaacctct ggcctctgtt cagaatgagt cccatgggat tccccggctg tgacactcta 6300 ccctccagaa cctgacgact gggccatgtg acccaaggag ggatccttac caagtgggtt 6360 ttcaccatcc tcttgctctc tgtctgagag atgttttcta aaccccagcc ttgaacttca 6420 ctcctccctc agtggtagtg tccaggtgcc gtggagcagc aggctggctt tggtaggggc 6480 acccatcacc cggcttgcct gtgcagtcag tgagtgctta gggcaaagag agctcccctg 6540 gttccattcc ttctgccacc caaaccctga tgagacctta gtgttctcca ggctctgtgg 6600 cccaggctga gagcagcagg gtagaaaaga ccaagatttg gggttttatc tctggttccc 6660 ttattactgc tctcaagcag tggcctctct cactttagcc atggaatggc tccgatctac 6720 ctcacagcag tgtcagaagg acttcgccag ggttttggga gctccagggt tcataagaag 6780 gtgaaccatt agaacagatc ccttcttttc cttttgcaat cagataaata aatatcactg 6840 aatgcagttc atcctcggcc ccctttccct ccgtttgttt tcttttcata atccacttac 6900 tcccttccct tctactctgc gctggctttt gacagaggca gtaaattagg cctaatcctc 6960 actcttttct tcctaatctt catcaaacaa aaaatgaaaa gtctgtctgg acgaagggga 7020 gtgagcttga gcctttgata tcttgctccc ccacccttcc tgaaactctt gaaatccagt 7080 tgccattgag tagcaaagcc acgctcccca caggacttgg acagagggcc cacaggggga 7140 tgggctggct gtggccaggt ttagggcagg gggcatttgt cccctccatg ctataatcca 7200 gtggtgcctt acatggtgtg tgtgtgtgtg tgtgcgtgtg tgtgtgtgtg tgtgtgtctg 7260 gaggcacagg cacaaagcat tgcttgggtt ggtcaaatgt cttgtgtcat aaatatattc 7320 tgatgtttcc cagcctttcc acaacctcta ccttcccact caccttcccc agctacaaaa 7380 atctgtatta tcctcttaaa gtaaaactgg agttacaaat ttgagcctgt tgatgcagtt 7440 ttctcttatt tccattttat gaatcttatc agccaggttt cctatgtaaa gaaaatttct 7500 aaggaaatca ttagatcatt ttgtgagttt ctatttaaac tctatgagcc aagactattg 7560 tccaggatga tttatggaat gcccggtaaa tcacagatgt tttgcgtgtt tgtgatgtag 7620 tgcgcaatgt gttgtcttgg ttcaggggcc ctagagttat aaaattccat ctgccccaag 7680 gaggagctcg atagatttcc acatatgggt gttccctgtg aattggacct ggtctgtgct 7740 gctctctttt ctccaggagc ccaggtttta tttctcagga tgttttcata ccaaagatga 7800 ctttctcctt ttcaaccagg gcagggtgag atctctggtg tctgaggtgt gcaagatttc 7860 ttggcaaaga ctcaactttg tggctgttag tggaggagga agtcttgctt gacaaataag 7920 aggccctcct ctccccatct ctggatattc tcccaacatc gctggataaa attggccagt 7980 cagtgctagg caggggagaa tgatgatgtt ggaggtgggg cccctggtgt gtagacttgt 8040 ccttgaacat tgtccagaag ctcatactgc ttgagtcttg ttggacttgc ccgtgcgcca 8100 gctgtcacac tcctttgcag tgtttagact tgtgctgggg ctatggattc catctgcaga 8160 ttgtcagata gaatctgaca gtctggtcat ttctccagca acttgtggcc aggcatcaag 8220 tggagggaga tttacatcct tggatgggag caggctgctt ttgctagcat gaggcctgag 8280 cacttgaact gtagaagaca aacagctgtc ccctgaaccc agagatatga tgacttccag 8340 gcccgtgaga acaacagagt ccctagagcc gctggcctcc ccagagtctg ttagctggag 8400 ctgggcatgg gacagaaatg acatggaaga gagggctgag ttggaggaga gggaatgaag 8460 agcgggcttt ttcagagctg acctaccctc tgcatcaagt ggaactgagg gggaacaggc 8520 agaatttggg tacaggagga tgtggggggc tgactttccc agactttctt ctggggaatg 8580 gaggtaggtg caggctgtgg tcctcacggg cagtccctgg gccttagtcc catgctgact 8640 gcatcctcaa ccctaggaga gtaggagcag ctcctctcct ctgtgtgttt ccattgcacc 8700 ctacatggtc ctaggagcac aggggaaggc atgggaatag cgcttcttac tgttttcatc 8760 ttaaaactgc ctagatccag gccttttgaa ggcctcttgg cgaaggtatg tctacacact 8820 gtctgtgttt gggtgtctgt ccagaacggg gaaatgggag cctgccatgg caagcactgc 8880 taatgctctc ccccacacaa caagccctgg gtatagtctg cctttggcca gtgagaagca 8940 gctgaagcat aacatgcaat tcagtttaat ttgaggccga ggtgggcgga ttacctgagg 9000 tcaggagttc gagaccagcc tggccaacat ggcaaaaccc cgtctctact aaaaatacaa 9060 aaattagccg ggcatggtgg tgggtgcctg taatcccagc tacttgggag gctgaggcag 9120 ggagaatgct tgaacccggg aggcagaggt tgcagtgagc ctagatggca ccactgcact 9180 ccagcctggg caacagagtg agactccatc tcaaaaaaaa aaaaaaaaaa aaaagggaca 9240 aggaaaaaga cagtctcata gaaatcaaat aacttccttg tatcgactct caccccatct 9300 accagattcc aaagtccatt ttctcttctc tatatgcact gcttagaagc agttgtgtga 9360 cactgaacaa gtcacttagc ctttctgact gtttctttgc ctgtgagtca actggagatt 9420 acatgatcac tctactcagc tcatgcaggc tgcttttgaa gacccggtga gacactgtgt 9480 gtgaaagtgg tttggaaact gaaaatgctt ctccggtata aagcggtgaa atcagaatct 9540 ggagcttgga tgagtcccag gtctattata gcagggatcc ccaacccctg ggccgcagac 9600 tgctactggt ctgtggcttg ttaggaactg ggctgcagcg caggaggtga gtggagggca 9660 agcgagcatt aacacgtgag ctccgcctcc tgtcagatct gcggctgcgt tagattctca 9720 ctggagcgcg aatcctaaag tgaactgcac atgtgaggga tctaggttgc atgctctttc 9780 tgagaatcta atgtctgata atctgaggtg gcagctttat cccaaaacca tctccttctc 9840 ccctgtccgt ggaaagattg ccttccacca aaccggtccc ttgtgccaaa aaggttgggg 9900 atcactgtct tatagggtct tgctctgttg cccaggctag agttcagggg cacaatcatg 9960 gcttattgca gcctcagcct cctgctcaag tgatcctctt atctcagcct cccaagtggc 10020 caggactaca ggcttgcacc actatgccca gctaattaaa ttttattttt tgtggagact 10080 gggtatttct atgttgccca ggctggtctt gagctcctgg gctcaagagt tcctcctgcc 10140 tcagcctccc aaagtgctgg gattatgagc atgatccact gtgcctggcc ctggttttgc 10200 tccctgtata ttcctgtttc tgtcgacagc ctgggtcttg gacccacttg tctttggtct 10260 ttgtggacat gccctgcctg gtctttcttc tgcaagtttg tactcacctc agactgactg 10320 ctttggtttt ctgtcctggt ctttatacat ctccacctct ggctctgttt ccatgttcag 10380 accctggtct ggcccgatat tttgctcctg gtctggatca ccccatcatt accaatgcaa 10440 catttacaac gagaacaatt ttaagaatgg gtaggccagg cgcagtggct cacgcctgta 10500 atcccagcac tttgggaggc cgaggcgggt ggatcacctg aggtcaggag ttcaagacca 10560 gcctggccaa catggtgaaa tcccatctct actaaaaata caaaagttag ccgggcgtgg 10620 tggcaggcgc ctgtaatcac agctgctcgg gaggctgagg caggagaatc gcttgaaccc 10680 agggaggcag aggttgcagt gagccgagat tgtgccactg cactctcaaa aaaaaaaaaa 10740 aaaaaaaaaa aagaatgaga agatttagaa cagaaggtac ttctggatct caggacatta 10800 tggtctatta tagtaggggg ttcatgagaa tgggacccta aggaaccaga aaggggagag 10860 aaggtggcgt atactttgct agggctgcca taacaaatga ctacaaactg ggtggcttaa 10920 cacaggagga actgattctc tcacagtgct ggagactaac agtccaaaat caaggtggca 10980 gcagggctgt gctccctgtg aggcgggagg ggaagatcct tccttgcctc ttctcttatc 11040 ccggtgtttg ctgacaatcc ttgggattgt ttgactggag ctgtatcact ctaacttctc 11100 ccccatcttc acaaggcctt cccctgtctg tctgtgccca catttctctc ctcttataag 11160 gacatcggcc attggatcag ggctcaacct aatccagcat gacctcattt gaacctggtt 11220 acatgtgcaa agaccccatt tccaaataag gtcacattca caagt 11265 <210> 3 <211> 472 <212> DNA <213> Homo sapiens <400> 3 cttgggtagt gacaaagtat taatttatta ggtgaagtat atgctttttt attagtgata 60 ataaatatat cctctctccc attataaaag tttgtatttc ttcttttaga aattgattct 120 tctgtcattt gcacatttat ctgtataatt ataacagggt atttcccagt ggtggctaat 180 gagagaatta tgggaaagta tagaacacta ttcaaatgca aagcactgta tgatttttat 240 ttaataggaa gacattttgt gcagcgattt ctgattgacc acagtttgat caagtgcatt 300 tgttaatgtg ttctacattt tcaaaaagga aaggagaatt tgttacattc agaacttgct 360 gccactcctt tgctacgtca taaagggtca gttgcccttg ctcatactga cctattcttt 420 accctctctg cttcttcttt gtgccagaag agtagaaatc tgaccctttg gg 472 <210> 4 <211> 25 <212> DNA <213> Artificial <220> <223> forward primer <400> 4 ggcaacccgg gaacggcagc gcctt 25 <210> 5 <211> 24 <212> DNA <213> Artificial <220> <223> forward primer <400> 5 gcaacccggg aacggcagcg cctt 24 <210> 6 <211> 23 <212> DNA <213> Artificial <220> <223> forward primer <400> 6 caacccggga acggcagcgc ctt 23 <210> 7 <211> 22 <212> DNA <213> Artificial <220> <223> forward primer <400> 7 aacccgggaa cggcagcgcc tt 22 <210> 8 <211> 21 <212> DNA <213> Artificial <220> <223> forward primer <400> 8 acccgggaac ggcagcgcct t 21 <210> 9 <211> 20 <212> DNA <213> Artificial <220> <223> forward primer <400> 9 cccgggaacg gcagcgcctt 20 <210> 10 <211> 19 <212> DNA <213> Artificial <220> <223> forward primer <400> 10 ccgggaacgg cagcgcctt 19 <210> 11 <211> 18 <212> DNA <213> Artificial <220> <223> forward primer <400> 11 cgggaacggc agcgcctt 18 <210> 12 <211> 31 <212> DNA <213> Artificial <220> <223> reverse primer <400> 12 ccaccaccca cacctcgtcc ctttsctgcg t 31 <210> 13 <211> 30 <212> DNA <213> Artificial <220> <223> reverse primer <400> 13 caccacccac acctcgtccc tttsctgcgt 30 <210> 14 <211> 29 <212> DNA <213> Artificial <220> <223> reverse primer <400> 14 accacccaca cctcgtccct ttsctgcgt 29 <210> 15 <211> 28 <212> DNA <213> Artificial <220> <223> reverse primer <400> 15 ccacccacac ctcgtccctt tsctgcgt 28 <210> 16 <211> 27 <212> DNA <213> Artificial <220> <223> reverse primer <400> 16 cacccacacc tcgtcccttt sctgcgt 27 <210> 17 <211> 26 <212> DNA <213> Artificial <220> <223> reverse primer <400> 17 acccacacct cgtcccttts ctgcgt 26 <210> 18 <211> 25 <212> DNA <213> Artificial <220> <223> reverse primer <400> 18 cccacacctc gtccctttsc tgcgt 25 <210> 19 <211> 24 <212> DNA <213> Artificial <220> <223> reverse primer <400> 19 ccacacctcg tccctttsct gcgt 24 <210> 20 <211> 23 <212> DNA <213> Artificial <220> <223> reverse primer <400> 20 cacacctcgt ccctttsctg cgt 23 <210> 21 <211> 22 <212> DNA <213> Artificial <220> <223> reverse primer <400> 21 acacctcgtc cctttsctgc gt 22 <210> 22 <211> 21 <212> DNA <213> Artificial <220> <223> reverse primer <400> 22 cacctcgtcc ctttsctgcg t 21 <210> 23 <211> 20 <212> DNA <213> Artificial <220> <223> forward primer <400> 23 ggccaccgtg ggaggcaacc 20 <210> 24 <211> 19 <212> DNA <213> Artificial <220> <223> forward primer <400> 24 gccaccgtgg gaggcaacc 19 <210> 25 <211> 18 <212> DNA <213> Artificial <220> <223> forward primer <400> 25 ccaccgtggg aggcaacc 18 <210> 26 <211> 17 <212> DNA <213> Artificial <220> <223> forward primer <400> 26 caccgtggga ggcaacc 17 <210> 27 <211> 16 <212> DNA <213> Artificial <220> <223> forward primer <400> 27 accgtgggag gcaacc 16 <210> 28 <211> 19 <212> DNA <213> Artificial <220> <223> reverse primer <400> 28 ggctgcggcc agcgaagtc 19 <210> 29 <211> 18 <212> DNA <213> Artificial <220> <223> reverse primer <400> 29 gctgcggcca gcgaagtc 18 <210> 30 <211> 17 <212> DNA <213> Artificial <220> <223> reverse primer <400> 30 ctgcggccag cgaagtc 17 <210> 31 <211> 16 <212> DNA <213> Artificial <220> <223> reverse primer <400> 31 tgcggccagc gaagtc 16 <210> 32 <211> 15 <212> DNA <213> Artificial <220> <223> reverse primer <400> 32 gcggccagcg aagtc 15 <210> 33 <211> 44 <212> DNA <213> Artificial <220> <223> forward primer <400> 33 ttatttaata ggaagacatt ttgtgcagcg atttctgatt gacc 44 <210> 34 <211> 43 <212> DNA <213> Artificial <220> <223> forward primer <400> 34 tatttaatag gaagacattt tgtgcagcga tttctgattg acc 43 <210> 35 <211> 42 <212> DNA <213> Artificial <220> <223> forward primer <400> 35 atttaatagg aagacatttt gtgcagcgat ttctgattga cc 42 <210> 36 <211> 41 <212> DNA <213> Artificial <220> <223> forward primer <400> 36 tttaatagga agacattttg tgcagcgatt tctgattgac c 41 <210> 37 <211> 40 <212> DNA <213> Artificial <220> <223> forward primer <400> 37 ttaataggaa gacattttgt gcagcgattt ctgattgacc 40 <210> 38 <211> 39 <212> DNA <213> Artificial <220> <223> forward primer <400> 38 taataggaag acattttgtg cagcgatttc tgattgacc 39 <210> 39 <211> 38 <212> DNA <213> Artificial <220> <223> forward primer <400> 39 aataggaaga cattttgtgc agcgatttct gattgacc 38 <210> 40 <211> 37 <212> DNA <213> Artificial <220> <223> forward primer <400> 40 ataggaagac attttgtgca gcgatttctg attgacc 37 <210> 41 <211> 36 <212> DNA <213> Artificial <220> <223> forward primer <400> 41 taggaagaca ttttgtgcag cgatttctga ttgacc 36 <210> 42 <211> 35 <212> DNA <213> Artificial <220> <223> forward primer <400> 42 aggaagacat tttgtgcagc gatttctgat tgacc 35 <210> 43 <211> 34 <212> DNA <213> Artificial <220> <223> forward primer <400> 43 ggaagacatt ttgtgcagcg atttctgatt gacc 34 <210> 44 <211> 33 <212> DNA <213> Artificial <220> <223> forward primer <400> 44 gaagacattt tgtgcagcga tttctgattg acc 33 <210> 45 <211> 32 <212> DNA <213> Artificial <220> <223> forward primer <400> 45 aagacatttt gtgcagcgat ttctgattga cc 32 <210> 46 <211> 31 <212> DNA <213> Artificial <220> <223> forward primer <400> 46 agacattttg tgcagcgatt tctgattgac c 31 <210> 47 <211> 30 <212> DNA <213> Artificial <220> <223> forward primer <400> 47 gacattttgt gcagcgattt ctgattgacc 30 <210> 48 <211> 29 <212> DNA <213> Artificial <220> <223> forward primer <400> 48 acattttgtg cagcgatttc tgattgacc 29 <210> 49 <211> 28 <212> DNA <213> Artificial <220> <223> forward primer <400> 49 cattttgtgc agcgatttct gattgacc 28 <210> 50 <211> 27 <212> DNA <213> Artificial <220> <223> forward primer <400> 50 attttgtgca gcgatttctg attgacc 27 <210> 51 <211> 26 <212> DNA <213> Artificial <220> <223> forward primer <400> 51 ttttgtgcag cgatttctga ttgacc 26 <210> 52 <211> 25 <212> DNA <213> Artificial <220> <223> forward primer <400> 52 tttgtgcagc gatttctgat tgacc 25 <210> 53 <211> 38 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (12) .. (12) <223> k stands for g or t <400> 53 ccctttatga cktagcaaag gagtggcagc aagttctg 38 <210> 54 <211> 37 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature &Lt; 222 > (11) <223> k stands for g or t <400> 54 cctttatgac ktagcaaagg agtggcagca agttctg 37 <210> 55 <211> 36 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (10) .. (10) <223> k stands for g or t <400> 55 ctttatgack tagcaaagga gtggcagcaa gttctg 36 <210> 56 <211> 35 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (9) .. (9) <223> k stands for g or t <400> 56 tttatgackt agcaaaggag tggcagcaag ttctg 35 <210> 57 <211> 34 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (8) .. (8) <223> k stands for g or t <400> 57 ttatgackta gcaaaggagt ggcagcaagt tctg 34 <210> 58 <211> 33 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (7) .. (7) <223> k stands for g or t <400> 58 tatgacktag caaaggagtg gcagcaagtt ctg 33 <210> 59 <211> 32 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature <222> (6) <223> k stands for g or t <400> 59 atgacktagc aaaggagtgg cagcaagttc tg 32 <210> 60 <211> 31 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature &Lt; 222 > (5) <223> k stands for g or t <400> 60 tgacktagca aaggagtggc agcaagttct g 31 <210> 61 <211> 30 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (4) .. (4) <223> k stands for g or t <400> 61 gacktagcaa aggagtggca gcaagttctg 30 <210> 62 <211> 29 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (3) .. (3) <223> k stands for g or t <400> 62 acktagcaaa ggagtggcag caagttctg 29 <210> 63 <211> 28 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature <222> (2) (2) <223> k stands for g or t <400> 63 cktagcaaag gagtggcagc aagttctg 28 <210> 64 <211> 27 <212> DNA <213> Artificial <220> <223> reverse primer <220> <221> misc_feature (222) (1) .. (1) <223> k stands for g or t <400> 64 ktagcaaagg agtggcagca agttctg 27 <210> 65 <211> 26 <212> DNA <213> Artificial <220> <223> reverse primer <400> 65 tagcaaagga gtggcagcaa gttctg 26 <210> 66 <211> 25 <212> DNA <213> Artificial <220> <223> reverse primer <400> 66 agcaaaggag tggcagcaag ttctg 25 <210> 67 <211> 24 <212> DNA <213> Artificial <220> <223> reverse primer <400> 67 gcaaaggagt ggcagcaagt tctg 24 <210> 68 <211> 23 <212> DNA <213> Artificial <220> <223> reverse primer <400> 68 caaaggagtg gcagcaagtt ctg 23 <210> 69 <211> 26 <212> DNA <213> Artificial <220> <223> probe <400> 69 cggcgcatgg cttcyattgg gtgcca 26 <210> 70 <211> 25 <212> DNA <213> Artificial <220> <223> probe <400> 70 ggcgcatggc ttcyattggg tgcca 25 <210> 71 <211> 24 <212> DNA <213> Artificial <220> <223> probe <400> 71 gcgcatggct tcyattgggt gcca 24 <210> 72 <211> 23 <212> DNA <213> Artificial <220> <223> probe <400> 72 cgcatggctt cyattgggtg cca 23 <210> 73 <211> 22 <212> DNA <213> Artificial <220> <223> probe <400> 73 gcatggcttc yattgggtgc ca 22 <210> 74 <211> 21 <212> DNA <213> Artificial <220> <223> probe <400> 74 catggcttcy attgggtgcc a 21 <210> 75 <211> 28 <212> DNA <213> Artificial <220> <223> probe <400> 75 ctggtcatcg tggccatcgc ccggactc 28 <210> 76 <211> 27 <212> DNA <213> Artificial <220> <223> probe <400> 76 tggtcatcgt ggccatcgcc cggactc 27 <210> 77 <211> 26 <212> DNA <213> Artificial <220> <223> probe <400> 77 ggtcatcgtg gccatcgccc ggactc 26 <210> 78 <211> 25 <212> DNA <213> Artificial <220> <223> probe <400> 78 gtcatcgtgg ccatcgcccg gactc 25 <210> 79 <211> 24 <212> DNA <213> Artificial <220> <223> probe <400> 79 tcatcgtggc catcgcccgg actc 24 <210> 80 <211> 23 <212> DNA <213> Artificial <220> <223> probe <400> 80 catcgtggcc atcgcccgga ctc 23 <210> 81 <211> 22 <212> DNA <213> Artificial <220> <223> probe <400> 81 atcgtggcca tcgcccggac tc 22 <210> 82 <211> 21 <212> DNA <213> Artificial <220> <223> probe <400> 82 tcgtggccat cgcccggact c 21 <210> 83 <211> 20 <212> DNA <213> Artificial <220> <223> probe <400> 83 cgtggccatc gcccggactc 20 <210> 84 <211> 19 <212> DNA <213> Artificial <220> <223> probe <400> 84 gtggccatcg cccggactc 19 <210> 85 <211> 18 <212> DNA <213> Artificial <220> <223> probe <400> 85 tggccatcgc ccggactc 18 <210> 86 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 86 ggccatcgcc cggactc 17 <210> 87 <211> 31 <212> DNA <213> Artificial <220> <223> probe <400> 87 cacagtttga tcgagtgcat ttgttaatgt g 31 <210> 88 <211> 30 <212> DNA <213> Artificial <220> <223> probe <400> 88 cacagtttga tcgagtgcat ttgttaatgt 30 <210> 89 <211> 29 <212> DNA <213> Artificial <220> <223> probe <400> 89 cacagtttga tcgagtgcat ttgttaatg 29 <210> 90 <211> 28 <212> DNA <213> Artificial <220> <223> probe <400> 90 cacagtttga tcgagtgcat ttgttaat 28 <210> 91 <211> 27 <212> DNA <213> Artificial <220> <223> probe <400> 91 cacagtttga tcgagtgcat ttgttaa 27 <210> 92 <211> 26 <212> DNA <213> Artificial <220> <223> probe <400> 92 cacagtttga tcgagtgcat ttgtta 26 <210> 93 <211> 25 <212> DNA <213> Artificial <220> <223> probe <400> 93 cacagtttga tcgagtgcat ttgtt 25 <210> 94 <211> 24 <212> DNA <213> Artificial <220> <223> probe <400> 94 cacagtttga tcgagtgcat ttgt 24 <210> 95 <211> 23 <212> DNA <213> Artificial <220> <223> probe <400> 95 cacagtttga tcgagtgcat ttg 23 <210> 96 <211> 22 <212> DNA <213> Artificial <220> <223> probe <400> 96 cacagtttga tcgagtgcat tt 22 <210> 97 <211> 21 <212> DNA <213> Artificial <220> <223> probe <400> 97 cacagtttga tcgagtgcat t 21 <210> 98 <211> 20 <212> DNA <213> Artificial <220> <223> probe <400> 98 cacagtttga tcgagtgcat 20 <210> 99 <211> 19 <212> DNA <213> Artificial <220> <223> probe <400> 99 cacagtttga tcgagtgca 19 <210> 100 <211> 18 <212> DNA <213> Artificial <220> <223> probe <400> 100 cacagtttga tcgagtgc 18 <210> 101 <211> 23 <212> DNA <213> Artificial <220> <223> probe <400> 101 cgcatggctt ccattgggtg cca 23 <210> 102 <211> 23 <212> DNA <213> Artificial <220> <223> probe <400> 102 cgcatggctt ctattgggtg cca 23 <210> 103 <211> 26 <212> DNA <213> Artificial <220> <223> forward primer <400> 103 gcaacccggg aacggcagcg ccttct 26 <210> 104 <211> 25 <212> DNA <213> Artificial <220> <223> forward primer <400> 104 caacccggga acggcagcgc cttct 25 <210> 105 <211> 24 <212> DNA <213> Artificial <220> <223> forward primer <400> 105 aacccgggaa cggcagcgcc ttct 24 <210> 106 <211> 23 <212> DNA <213> Artificial <220> <223> forward primer <400> 106 acccgggaac ggcagcgcct tct 23 <210> 107 <211> 22 <212> DNA <213> Artificial <220> <223> forward primer <400> 107 cccgggaacg gcagcgcctt ct 22 <210> 108 <211> 21 <212> DNA <213> Artificial <220> <223> forward primer <400> 108 ccgggaacgg cagcgccttc t 21 <210> 109 <211> 20 <212> DNA <213> Artificial <220> <223> forward primer <400> 109 cgggaacggc agcgccttct 20 <210> 110 <211> 19 <212> DNA <213> Artificial <220> <223> forward primer <400> 110 gggaacggca gcgccttct 19 <210> 111 <211> 18 <212> DNA <213> Artificial <220> <223> forward primer <400> 111 ggaacggcag cgccttct 18 <210> 112 <211> 19 <212> DNA <213> Artificial <220> <223> probe <400> 112 cccaatrgaa gccatgcgc 19 <210> 113 <211> 18 <212> DNA <213> Artificial <220> <223> probe <400> 113 cccaatrgaa gccatgcg 18 <210> 114 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 114 cccaatrgaa gccatgc 17 <210> 115 <211> 16 <212> DNA <213> Artificial <220> <223> probe <400> 115 cccaatrgaa gccatg 16 <210> 116 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 116 cccaatrgaa gccat 15 <210> 117 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 117 ctcggagtcc gggcgat 17 <210> 118 <211> 16 <212> DNA <213> Artificial <220> <223> probe <400> 118 ctcggagtcc gggcga 16 <210> 119 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 119 ctcggagtcc gggcg 15 <210> 120 <211> 14 <212> DNA <213> Artificial <220> <223> probe <400> 120 ctcggagtcc gggc 14 <210> 121 <211> 13 <212> DNA <213> Artificial <220> <223> probe <400> 121 ctcggagtcc ggg 13 <210> 122 <211> 12 <212> DNA <213> Artificial <220> <223> probe <400> 122 ctcggagtcc gg 12 <210> 123 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 123 ctcggagtcc aggcgat 17 <210> 124 <211> 16 <212> DNA <213> Artificial <220> <223> probe <400> 124 ctcggagtcc aggcga 16 <210> 125 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 125 ctcggagtcc aggcg 15 <210> 126 <211> 14 <212> DNA <213> Artificial <220> <223> probe <400> 126 ctcggagtcc aggc 14 <210> 127 <211> 13 <212> DNA <213> Artificial <220> <223> probe <400> 127 ctcggagtcc agg 13 <210> 128 <211> 12 <212> DNA <213> Artificial <220> <223> probe <400> 128 ctcggagtcc ag 12 <210> 129 <211> 27 <212> DNA <213> Artificial <220> <223> primer <400> 129 catgcccacc acccacacct cgtccct 27 <210> 130 <211> 26 <212> DNA <213> Artificial <220> <223> primer <400> 130 atgcccacca cccacacctc gtccct 26 <210> 131 <211> 25 <212> DNA <213> Artificial <220> <223> primer <400> 131 tgcccaccac ccacacctcg tccct 25 <210> 132 <211> 24 <212> DNA <213> Artificial <220> <223> primer <400> 132 gcccaccacc cacacctcgt ccct 24 <210> 133 <211> 23 <212> DNA <213> Artificial <220> <223> primer <133> 133 cccaccaccc acacctcgtc cct 23 <210> 134 <211> 22 <212> DNA <213> Artificial <220> <223> primer <400> 134 ccaccaccca cacctcgtcc ct 22 <210> 135 <211> 21 <212> DNA <213> Artificial <220> <223> primer <400> 135 caccacccac acctcgtccc t 21 <210> 136 <211> 20 <212> DNA <213> Artificial <220> <223> primer <400> 136 accacccaca cctcgtccct 20 <210> 137 <211> 19 <212> DNA <213> Artificial <220> <223> primer <400> 137 ccacccacac ctcgtccct 19 <210> 138 <211> 18 <212> DNA <213> Artificial <220> <223> primer <400> 138 cacccacacc tcgtccct 18 <139> <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 139 cacagtttga tcgagtg 17 <210> 140 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 140 cacccaatgg aagcc 15 <210> 141 <211> 16 <212> DNA <213> Artificial <220> <223> probe <400> 141 ccaatggaag ccatgc 16 <210> 142 <211> 20 <212> DNA <213> Artificial <220> <223> probe <400> 142 catggcttcc attgggtgcc 20 <210> 143 <211> 18 <212> DNA <213> Artificial <220> <223> probe <400> 143 catggcttcc attgggtg 18 <210> 144 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 144 ggcttccatt gggtgcc 17 <210> 145 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 145 ggcacccaat agaagcc 17 <210> 146 <211> 20 <212> DNA <213> Artificial <220> <223> probe <400> 146 ccggcgcatg gcttccattg 20 <210> 147 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 147 tagaagccat gcgcc 15 <210> 148 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 148 ccaatagaag ccatgcg 17 <210> 149 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 149 ccaatggaag ccatg 15 <210> 150 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 150 tggaagccat gcgcc 15 <210> 151 <211> 15 <212> DNA <213> Artificial <220> <223> probe <400> 151 caatggaagc catgc 15 <210> 152 <211> 17 <212> DNA <213> Artificial <220> <223> probe <400> 152 cttccattgg gtgccag 17 <210> 153 <211> 16 <212> DNA <213> Artificial <220> <223> probe <400> 153 ccattgggtg ccagca 16 <210> 154 <211> 22 <212> DNA <213> Artificial <220> <223> probe <400> 154 cttccattgg gtgccagcaa ga 22 <210> 155 <211> 20 <212> DNA <213> Artificial <220> <223> probe <400> 155 gctggcaccc aatggaagcc 20 <210> 156 <211> 18 <212> DNA <213> Artificial <220> <223> probe <400> 156 ccaatggaag ccatgcgc 18 <210> 157 <211> 20 <212> DNA <213> Artificial <220> <223> probe <400> 157 ccaatggaag ccatgcgccg 20 <210> 158 <211> 19 <212> DNA <213> Artificial <220> <223> probe <400> 158 ccaatggaag ccatgcgcc 19 <210> 159 <211> 18 <212> DNA <213> Artificial <220> <223> probe <400> 159 ggcttccatt gggtgcca 18 <210> 160 <211> 22 <212> DNA <213> Artificial <220> <223> probe <400> 160 catggcttcc attgggtgcc ag 22 <210> 161 <211> 40 <212> DNA <213> Artificial <220> <223> DNA <400> 161 cgccttcttg ctggcaccca atagaagcca tgcgccggac 40 <210> 162 <211> 40 <212> DNA <213> Artificial <220> <223> DNA <400> 162 cgccttcttg ctggcaccca atggaagcca tgcgccggac 40 <210> 163 <211> 40 <212> DNA <213> Artificial <220> <223> DNA <400> 163 gtccggcgca tggcttctat tgggtgccag caagaaggcg 40 <210> 164 <211> 40 <212> DNA <213> Artificial <220> <223> DNA <400> 164 gtccggcgca tggcttccat tgggtgccag caagaaggcg 40 <210> 165 <211> 23 <212> DNA <213> Artificial <220> <223> primer <400> 165 gggaacggca gcgccttctt gct 23 <210> 166 <211> 26 <212> DNA <213> Artificial <220> <223> primer <400> 166 tggtgaccgt ctgcagacgc tcgaac 26  

Claims (43)

As a primer set for amplifying obesity genes simultaneously in the same reaction solution by gene amplification, The obesity gene is β2AR gene, β3AR gene and UCP1 gene, A primer set for obesity gene amplification, comprising the following primer sets (1) to (3): Primer Set (1) A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F1) and a reverse primer consisting of an oligonucleotide of (R1) (F1) at least one oligonucleotide having the same sequence as the region from the 4248th thymine base (T) in the nucleotide sequence of SEQ ID NO: 1 to the 18-25 base in the 5 'direction, as the first base; An oligonucleotide having the thymine base (T) at the 3 'end, and At least one oligonucleotide having the same sequence as the region from the 18th to the 26th base in the 5 'direction with the 4250th thymine base (T) as the first base in the nucleotide sequence of SEQ ID NO: 1; Oligonucleotides with 3 'Terminus (T) At least one oligonucleotide (R1) at least one oligonucleotide complementary to a region from the 21st to 31th base in the 3 'direction with the 4292th adenine base (A) in the nucleotide sequence of SEQ ID NO: 1 as the first base; An oligonucleotide having a 3 'end of thymine base (T) complementary to the 4292th adenine base (A), and At least one oligonucleotide complementary to the region from the 18th to the 27th base in the 3 'direction with the 4301th adenine base (A) in the nucleotide sequence of SEQ ID NO: 1 as the first base, wherein the 4301st Oligonucleotide having 3 'end of thymine base (T) complementary to adenine base (A) At least one oligonucleotide, Primer Set (2) A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F2) and a reverse primer consisting of an oligonucleotide of (R2) (F2) at least one oligonucleotide having the same sequence as the region from the 4110's cytosine base (C) in the nucleotide sequence of SEQ ID NO: 2 as the 16th to 20th base in the 5 'direction, as the 1st base; Oligonucleotide having said cytosine base (C) as 3 'end (R2) at least one oligonucleotide complementary to a region from the 15th to 19th base in the 3 'direction with the 4172nd guanine base (G) in the nucleotide sequence of SEQ ID NO: 2 as the first base; An oligonucleotide having the 3 'end of a cytosine base (C) complementary to the 4172 th guanine base (G), Primer Set (3) A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F3) and a reverse primer consisting of an oligonucleotide of (R3) (F3) at least one oligonucleotide having the same sequence as the region from the 25th to the 44th base in the 5 'direction with the 280th cytosine base (C) as the first base in the nucleotide sequence of SEQ ID NO: 3, Oligonucleotide having said cytosine base (C) as 3 'end (R3) at least one oligonucleotide complementary to the region from the 23rd to the 38th base in the 3 'direction with the 350th cytosine base (C) as the first base in the nucleotide sequence of SEQ ID NO: 3, An oligonucleotide having 3 'terminus of guanine base (G) complementary to the 350th cytosine base (C). The primer set according to claim 1, wherein the primer sets of (1) to (3) are primer sets of the following (1 ') to (3'), respectively: Primer set (1 ') A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F1 ') and a reverse primer consisting of an oligonucleotide of (R1') (F1 ′) at least one oligonucleotide of an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 9 and an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 109; (R1 ') at least one oligonucleotide selected from the group consisting of an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 12, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 18, and an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 134, Primer set (2 ') A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F2 ') and a reverse primer consisting of an oligonucleotide of (R2') (F2 ') at least one oligonucleotide among oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 24 and oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 25 (R2 ') at least one oligonucleotide of the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 28, and the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 30, Primer set (3 ') A pair of primer sets comprising a forward primer consisting of an oligonucleotide of (F3 ') and a reverse primer consisting of an oligonucleotide of (R3') (F3 ') oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 43 (R3 ') Oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 63. The primer set for obesity gene amplification according to claim 1, wherein said primer set for obesity gene amplification is a primer set for amplifying obesity gene in a biological sample. The primer set for the amplification of obesity genes according to claim 3, wherein the biological sample is whole blood. As a reagent for amplifying obesity genes simultaneously in the same reaction solution by gene amplification, The obesity gene is a β2AR gene, a β3AR gene, and a UCP1 gene, and the obesity gene amplification reagent comprising the primer set for amplification of the obesity gene according to claim 1. The method of claim 5, further comprising a probe capable of hybridizing to the detection target site of the β2AR gene, a probe capable of hybridizing to the detection target site of the β3AR gene, and a probe capable of hybridizing to the detection target site of the UCP1 gene. Obesity gene amplification reagent containing. The method for amplifying obesity genes according to claim 6, wherein the probe is a probe comprising an oligonucleotide of (P1 '), a probe consisting of an oligonucleotide of (P2'), and an oligonucleotide of (P3 '). reagent: (P1 ') at least one oligonucleotide of the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 72, and the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 114, (P2 ') at least one oligonucleotide selected from the group consisting of an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 83, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 120, and an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 127, (P3 ') At least one oligonucleotide among the oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 95 and the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 139. The reagent of claim 6, wherein the probe is a fluorescent labeled probe. As a method of producing amplification products of obesity genes simultaneously in the same reaction solution by gene amplification, The obesity gene is β2AR gene, β3AR gene and UCP1 gene, A process for producing an amplification product, comprising the following step (I): (I) A step of amplifying the β2AR gene, β3AR gene, and UCP1 gene in a reaction solution by using the nucleic acid in the sample as a template and using the primer set for the obesity gene amplification according to claim 1. 10. The method according to claim 9, wherein in the step (I), the probe is capable of hybridizing to a detection target site of the β2AR gene, a probe capable of hybridizing to a detection target site of the β3AR gene and a UCP1 gene in the reaction solution. A method of producing an amplification product, further comprising adding a hybridizable probe to the site of interest. The method of claim 10, wherein the probe is a probe consisting of an oligonucleotide of (P1 ′), a probe consisting of an oligonucleotide of (P2 ′), and a probe consisting of an oligonucleotide of (P3 ′). (P1 ') at least one oligonucleotide of the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 72, and the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 114, (P2 ') at least one oligonucleotide selected from the group consisting of an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 83, an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 120, and an oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 127, (P3 ') At least one oligonucleotide among the oligonucleotides consisting of the nucleotide sequence of SEQ ID NO: 95 and the oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 139. The method of claim 10, wherein the probe is a fluorescent labeled probe. The method of claim 12, further comprising the following step (II): (II) A step of measuring the fluorescence intensity of the fluorescent label in the fluorescent labeling probe with respect to the reaction solution. The method of claim 9, wherein the sample is a biological sample. The method of claim 14, wherein the biological sample is whole blood. The method for producing an amplified product according to claim 15, wherein an addition ratio of the whole blood sample in the reaction solution is 0.1 to 0.5% by volume. As a method of analyzing the polymorphism of the detection target site in the obesity gene, The obesity gene is a β2AR gene, a β3AR gene, and a UCP1 gene, and includes the following steps (i) to (iv): (Iii) amplifying each region including the detection target site in the β2AR gene, β3AR gene, and UCP1 gene in the reaction solution by the method for producing an amplification product according to claim 9, (Ii) the amplification product in step (iii), a probe capable of hybridizing to the site of detection of the β2AR gene, a probe capable of hybridization to the site of detection of the β3AR gene, and a site of detection of the UCP1 gene Preparing a reaction solution containing a redistributable probe, (Iii) changing a temperature of the reaction solution and measuring a signal value indicating a state of melting of the hybridization product between the amplification product and the probe, (Iii) A step of determining the polymorphism of each detection target site in the β2AR gene, β3AR gene, and UCP1 gene from the change in the signal value due to the temperature change. 18. The probe according to claim 17, wherein in the step (iii), a probe capable of hybridizing to a detection target site of the β2AR gene and a probe capable of hybridizing to a detection target site of the β3AR gene prior to the amplification reaction. And a polymorphic analysis method in which a hybridizable probe is added to a detection target site of the UCP1 gene. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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